Original Article
The p27 Pathway Modulates The Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone-Related Peptide† Min Zhu1*, Jing Zhang2*, Zhan Dong1, Ying Zhang1, Rong Wang1, Andrew Karaplis3, David Goltzman4, and Dengshun Miao1 1
State Key Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells, Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing 210029, Jiangsu, People's Republic of China; 2Department of Human Anatomy, Basic Medical College of Nanchang University, Nanchang 330046, Jiangxi, People's Republic of China; 3Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Canada; 4Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada * These authors contributed equally to this work. Corresponding author: Dengshun Miao, M.D., Ph.D. State Key Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells Nanjing Medical University, Nanjing, Jiangsu, 210029, The People’s Republic of China Tel & FAX: 011-86-25-8686-2015 E-mail: [email protected]
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2544] Additional Supporting Information may be found in the online version of this article. Initial Date Submitted December 16, 2014; Date Revision Submitted April 15, 2015; Date Final Disposition Set April 23, 2015
Journal of Bone and Mineral Research This article is protected by copyright. All rights reserved DOI 10.1002/jbmr.2544
This article is protected by copyright. All rights reserved
1
Abstract Parathyroid hormone–related peptide (PTHrP) 1–84 knock-in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild type (WT) littermates. We found that the expression levels of p27, p16 and p53 were significantly up-regulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice which were homozygous for both p27 deletion and the Pthrp KI mutation (p27-/-Pthrp KI). We then compared p27-/-Pthrp KI mice with p27-/-, Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of PCNA positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and ALP-, type I collagen- and osteocalcin-positive bone areas were increased in p27-/- mice, and reduced in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however these parameters were increased in p27-/-Pthrp KI mice compared to Pthrp KI mice. As well, protein expression levels of PTHR, IGF-1 and Bmi-1, and the numbers of total CFU-f and ALP positive CFU-f were similarly increased in p27-/-Pthrp KI mice compared to Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the p27 pathway may function downstream in the action of PTHrP to regulate skeletal growth and development. This article is protected by copyright. All rights reserved Keywords：PTHrP, p27, skeletal growth, osteoblastic bone formation.
This article is protected by copyright. All rights reserved
2
Introduction Parathyroid hormone-related peptide (PTHrP), which was first identified in cancers associated with humoral hypercalcemia (1), is a pleiotropic factor with multiple physiological functions, including the regulation of morphogenesis, cell proliferation and differentiation, and transplacental calcium transport (2,3). PTHrP is a polyhormone which can be translated and processed into several smaller bioactive forms. The N-terminal region of PTHrP, i.e., PTHrP (1–36), mediates ‘‘classical’’ PTH-like effects. The midregion, i.e., PTHrP (37–86), has been implicated in placental calcium transport (4), and PTHrP (108–139), has been reported to inhibit bone resorption (5). A bipartite nuclear localization signal (NLS) is located in PTHrP (87–107) and can translocate heterologous plasma proteins into the nucleolus (6).
To examine the functional consequences of nuclear localization of PTHrP in vivo, we had previously generated a mouse expressing a partial ablation of Pthrp, that is, a Pthrp “knockin (KI) mouse”. Pthrp KI mice express PTHrP (1–84), a truncated form of PTHrP that is missing the NLS and the C-terminal region but can still signal through its cell surface receptor (7). Pthrp KI mice exhibit retarded growth, skeletal dysplasia and early senescence, leading to a rapid demise after birth (7). These results suggested that the NLS and C terminus of PTHrP play a critical role in bone development and function by stimulating the proliferation and differentiation of bone marrow cells. Other in vivo genetic mouse models have also indicated that the mid-region and C-terminus of PTHrP are functional (8).
Cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves
cyclin
dependent
kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different This article is protected by copyright. All rights reserved
3
phases
of the cell
cycle.
One CKI of importance is p27KIP1. The p27KIP1 protein was first identified
as an inhibitor of cyclin E/CDK2 complexes during TGFβ-induced G1 arrest (9). The p27KIP1 protein inhibits cyclin/CDK activity by binding cyclin/CDK complexes through its N-terminal domain, thus blocking ATP binding, and physically occluding the catalytic cleft of the CDK(10)，(11,12).
The p27KIP1 null (p27-/-) mouse shows an overall increase in cell proliferation, resulting in approximately 30% increase in body size and multiple organ hyperplasia (13-15). Bone marrow cells from p27 null mice exhibited increased proliferative activity and form an increased number and larger size of osteoblastic colonies, which can further differentiate to the stage of mineralization (16). PTHrP induces proliferation in arterial smooth muscle cells via a PTHrP/p27KIP1 pathway in which PTHrP-induces proteasomal degradation of p27 (17). In contrast, mutant PTHrP devoid of the NLS, markedly inhibits arterial smooth muscle cell cycle and neointima formation by up-regulation of p27KIP1 (18). However, it is unclear whether the NLS and C terminus of PTHrP regulate bone formation via a p27KIP1 pathway. In this work, we generated compound mutant mice which are homozygous for both p27 deletion and the PTHrP KI mutation (p27 -/- PTHrP KI), and compared their phenotypes with those of p27 -/-, PTHrP KI and WT littermates.
This article is protected by copyright. All rights reserved
4
Materials and Methods Derivation and genotyping of mice The p27-/- mice (C57BL/6J hybrid background) (13) used in this study were purchased from the Jackson Laboratory. The Pthrp KI mice (C57BL/6J hybrid background) used in this study were generated and characterized as we previously described (7). p27+/- mice and Pthrp KI+/- mice were fertile and were mated to produce offspring heterozygous at both loci, which were then mated to generate p27-/-Pthrp KI-/- pups. All animal experiments were carried out in compliance with, and approval by, the Institutional Animal Care and Use Committee. The genotype of p27-/- and Pthrp KI mice was confirmed as described previously (7,19).
Microarray analysis and real-time RT-PCR RNA was isolated from mouse bone tissue, excluding the growth plate, using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. Microarray analysis and time RT-PCR were performed as described previously (20,21) and as described in detail in the Supplementary Materials and Methods.
Iconographic and histopathological analyses Femurs or tibiae were removed and analyzed by radiography, micro-computed tomography, histology, histochemistry and immunohistochemistry as we described previously (21).
Western blot analysis Proteins were extracted from bone tissue and immunoblotting was carried out as we described previously (22) and as described in detail in the Supplementary Materials and Methods.
This article is protected by copyright. All rights reserved
5
CFU-f assay and cytochemical staining Bone marrow cells were flushed out from tibiae and femurs of 2-week-old mice and 106 bone marrow cells were cultured in 36-cm2 petri dishes for 10 to 18 days. At the end of the culture period, cells were stained with methyl blue or cytochemically for ALP. Following staining, the positive areas of colony-forming units, fibroblastic (CFU-f) and ALP positive CFU-f (CFU-fap) were measured using computer-assisted image analysis as we described previously (23).
Statistical analysis Data from image analysis are presented as mean ± s.e.m. Statistical comparisons were made using a two-way ANOVA, with P2 fold up- or down-regulated genes, p < 0.05) (Supplementary Table 1), in the bone tissue of E18.5 Pthrp KI mice compared with their WT littermates; 11 genes (listed in Table 1) were related to BM-MSC differentiation and cell cycle control. The most dramatically down-regulated genes in Pthrp KI mice included V-ATPase, osterix, cyclin D1, cyclin E, CDK2, ALP and Runx2. In contrast, the expression of 4 genes, p16, p53, p27 and PPARγ2 were significantly up-regulated. Fewer genes were found to be differentially expressed based on the q-values than on the p-values. The expression levels (log scale) of the genes related to BM-MSC differentiation and cell cycle control were reordered and displayed in a heat map, with the spectrum ranging from green (low level) to red (high level), as presented in Fig. 1A. The genes related to BM-MSC differentiation included osterix, ALP, Runx2 and PPARγ2. The genes related to cell cycle control included cyclin D1, cyclin E, CDK2, p16, p53 and p27. We validated the microarray analysis data using real-time PCR. This was carried out using gene-specific primers and the same RNA sources used for the microarray analysis. The results found by real-time PCR corroborated those from the microarray analysis, as shown in Fig.1B. The genes that were significantly different in Pthrp KI mice and WT mice showed similar alterations in expression with both measurement techniques.
Previous in vitro studies have implicated the PTHrP nuclear localization signal in inducing proliferation in arterial smooth muscle cells by modulating p27 (17). In view of our finding of upregulated p27 in skeletal tissue of Pthrp KI mice we next determined whether the cell cycle inhibitor p27Kip1 was involved in the regulation by PTHrP of skeletal growth and development in This article is protected by copyright. All rights reserved
7
vivo. We therefore generated compound mutant mice which are homozygous for both p27 deletion and the Pthrp KI mutation (p27-/-Pthrp KI), and compared them with p27-/-, Pthrp KI, and WT littermates.
The effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice To determine whether the deletion of p27 can rescue the growth retardation and premature senescence caused by Pthrp KI, we examined the effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice. Our results showed that p27-/- mice and WT mice survived for more than 2 years in contrast to the mean survival time of about 2 weeks in Pthrp KI mice; deletion of p27 in the Pthrp KI mice prolonged survival in the p27-/-Pthrp KI mice to about 3 weeks (Fig. 1C). The body weight and habitus were increased significantly in p27-/- mice, but were reduced in both Pthrp KI and p27-/-Pthrp KI mice compared to their WT littermates; however they were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 1D-F).
The effect of deletion of p27 on the skeletal growth of Pthrp KI mice To assess whether the deletion of p27 can correct skeletal growth retardation observed in the Pthrp KI, we examined the effect of deletion of p27 on the length of long bones and the proliferation of chondrocytes of Pthrp KI mice. We found that the length of long bones, the width of the proliferating zone of cartilage growth plates and the percentage of PCNA positive chondrocytes were clearly increased in p27-/- mice, but were reduced significantly in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; nevertheless these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 2A-G).
The effect of deletion of p27 on bone volume and osteoblastic bone formation of Pthrp KI mice This article is protected by copyright. All rights reserved
8
To determine whether the deletion of p27 can correct premature osteoporosis caused by Pthrp KI, we examined the effect of deletion of p27 on bone volume and osteoblastic indices in Pthrp KI mice. BMD (Figs. 2A and C), epiphyseal, trabecular and cortical bone volume (Figs. 3A-F), osteoblast numbers (Figs. 4A and B), and ALP (Figs. 4C and D), and type I collagen (Col I) (Figs. 4E and F) and osteocalcin (OCN) (Figs. 4G and H) positive areas, and the percentage of Bmi1 positive osteoblasts relative to total osteoblasts were increased significantly in p27-/- mice, but were reduced significantly in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice.
The effect of deletion of p27 on the expression of osteogenesis-regulating molecules of Pthrp KI mice To further investigate whether the effect of deletion of p27 on osteoblastic activity of Pthrp KI mice is associated with alterations of expression levels of osteogenesis regulating molecules, we examined the protein expression levels in bone tissue of IGF-1, the type I PTH/PTHrP receptor (PTHR), protooncogene Bmi-1 and cell cyclin-dependent kinase inhibitors p16 and p53. The results showed that protein expression levels of IGF-1, PTHR and Bmi-1 were up-regulated in p27-/- mice, but were down-regulated in both Pthrp KI and p27-/- Pthrp KI mice compared to wild-type mice, however they were up-regulated in p27-/-Pthrp KI mice relative to Pthrp KI mice (Figs. 5A-D). We have previously reported that protein expression of PTHR in situ correlates with alkaline phosphatase staining of osteoblasts and quantitation of osteoblastic perimeter per bone perimeter (Ob.Pm/B.Pm) as well as with serum levels of osteocalcin (24,25). Consequently the altered expression levels of PTHR likely reflected changes in the number of osteoblastic cells that were observed. In contrast, protein expression levels of p53 and p16 were up-regulated in all three mutant models ie p27-/-, Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however they were up-regulated This article is protected by copyright. All rights reserved
9
more in Pthrp KI mice compared to p27-/-Pthrp KI mice (Figs. 5A, E and F).
The effect of deletion of p27 on the proliferation of BM-MSCs and differentiation into osteoblasts of Pthrp KI mice To assess whether the effect of deletion of p27 on osteoblasts of Pthrp KI mice was associated with alterations of the recruitment and differentiation of BM-MSCs, CFU-f assays were performed and the alterations in expression of genes related to osteogenic cell differentiation were determined. We found that CFU-f and ALP positive area and the gene expression levels of Runx2, ALP, type I collagen, osteocalcin and Bmi1 were increased significantly in p27-/- mice compared to WT mice, but were decreased significantly in Pthrp KI mice compared to WT mice. However, these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 6A-I). We also examined the expression levels of mRNA encoding the N-terminal domain of PTHrP and encoding PTHR in the osteogenic cells resulting from 10 day BM-MSC cultures. Results revealed that mRNA expression level of the N-terminus of PTHrP (Pthlh) was increased significantly in p27-/- mice, but was not altered significantly in Pthrp KI and p27-/-Pthrp KI mice compared to WT mice (Fig. 6J). The mRNA expression level of PTHR was increased significantly in p27-/- mice and decreased significantly in Pthrp KI mice, however, it was not altered in p27-/-Pthrp KI mice compared to WT mice (Fig. 6K).
The effect of deletion of p27 on osteoclastic bone resorption of Pthrp KI mice To investigate whether the effect of deletion of p27 on the alteration of bone volume of Pthrp KI mice is associated with the alteration of osteoclastic bone resorption, we examined the effect of deletion of p27 on osteoclastic bone resorption of Pthrp KI mice. The results showed that the ratio of RANKL/OPG and the TRAP positive osteoclast surface were increased in p27-/- mice, but were This article is protected by copyright. All rights reserved
10
decreased significantly in Pthrp KI and p27-/-Pthrp KI mice compared to WT mice. However, these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 6K-M).
This article is protected by copyright. All rights reserved
11
Discussion In this study, we used a genetic approach to gain insight into the role of p27 in contributing to the skeletal phenotype of Pthrp KI mice and to determine whether p27 might function downstream of PTHrP NLS and C-terminus in modifying bone formation and development.
Pthrp KI mice showed growth retardation and muscle atrophy, hyperkeratosis, body fat reduction and poor bone development as well as other phenotypes (7). It has recently been reported that although Pthrp KI mice exhibit defects in mammary mesenchyme differentiation and mammary duct outgrowth, the mammary buds in PTHrP (1-84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function (26). To address this issue we examined expression of mRNA encoding N-terminal PTHrP in osteoblastic cells derived from BMSCs from Pthrp KI mice as well as from WT and the other mutants used in our studies and found that expression levels of mRNA encoding N-terminal PTHrP were not significantly decreased in Pthrp KI osteoblastic cells. Our results are consistent with our previous observations in embryonic fibroblasts, suggesting that the osteogenesis defects occurring in Pthrp KI mice resulted from the loss of PTHrP nuclear function (7).
Cyclin-dependent kinase inhibitors are an important class of negative regulators of cell cycle proteins, of which p27 is a key member. P27 functions primarily by binding to cyclin-dependent protein kinases or cyclin-CDK complexes, to achieve cyclin-dependent protein kinase activity inhibition, thereby negatively regulating the cell cycle (27). Previous studies have shown that p27 knockout mice showed accelerated growth, and multi-organ hypertrophy due to increased cell proliferation (13,28). We found, via our microarray analysis, that p27 is a direct skeletal target of This article is protected by copyright. All rights reserved
12
PTHrP in vivo and that deletion of p27 in Pthrp KI mice partially rescued the phenotype of the growth retardation of Pthrp KI mice. Osteoblastic cells differentiate in bone marrow from BM-MSCs, which are pluripotent stem cells having the potential to differentiate into a variety of cell lineages. Drissi et al found that the ability of BM-MSCs to progress along the osteoblastic lineage was significantly higher in BM-MSCs derived from p27-/ - mice compared to those from with WT mice (16). Our data confirmed the above results and also showed that increased osteoblastic differentiation of BM-MSCs occurs when p27 is deleted from Pthrp KI mice as compared to BM-MSCs derived from Pthrp KI mice. Consequently p27 appears to lie downstream of the PTHrP NLS and C-terminus in stimulating osteoblastic differentiation from BM-MSCs. Fiaschi-Taesch et al found that specific mutations of PTHrP NLS can increase the expression levels of p27, such that vascular smooth muscle cells undergo growth arrest in vitro (18). Consequently PTHrP inhibition of the expression of p27 can promote the proliferation of vascular smooth muscle (17). Our studies show that a similar mechanism may occur in vivo with respect to skeletal growth and development and osteoblastic bone formation. Nevertheless a limitation of our study is that the use of a global knockout of p27 and of a model of global ablation of the NLS and C-terminus of PTHrP does not preclude the fact that indirect regulation of PTHrP action may still occur in the p27-/-Pthrp KI mouse model we have used. Consequently targeted gene ablation in skeletal cells would be important to do in future studies.
In vivo, compared with 2-week-old Pthrp KI mice, the weight, long bone length, width of the growth plate, chondrocyte proliferation percentage, bone mineral density, bone volume, osteoblastic bone formation and osteoclastic bone resorption parameters
were all increased significantly in
p27-/-Pthrp KI mice. P27 was first identified as a CDK inhibitors due to its ability to block the activity of cyclin E/ CDK2 and cyclin A/ CDK2 in cells arrested in G1 by lovastatin, TGF-β and This article is protected by copyright. All rights reserved
13
contact inhibition (29-31). Our results demonstrated that mRNA expression levels of cyclin E and CDK2 were down-regulated significantly, whereas mRNA expression levels of p27 were up-regulated significantly in Pthrp KI mouse bone. These results suggest that deletion of p27 improved skeletal growth and development in Pthrp KI mice is, at least partially, mediated by up-regulating cyclin E and CDK2. We also found that the expression of Bmi-1 at both gene and protein levels and Bmi1 positive osteoblasts were increased in p27-/- mouse bone, but were decreased in Pthrp KI mouse bone and increased in Pthrp KI mouse bone by deletion of p27. However, it remains to be investigated how p27 regulates Bmi-1.
Nevertheless the deletion of p27 in Pthrp KI mice only modestly, although significantly, improved the Pthrp KI mouse skeletal dysplasia phenotype (32);
consequently it is likely that other
mechanisms, beside p27 are driving the Pthrp KI phenotype and that other genes lie downstream of the PTHrP NLS and C-terminus in modifying skeletal growth and development..Previous studies have shown that p16 can selectively inhibit the activities of cyclin D:CDK4/6 complexes, preventing phosphorylation by pRb, and causing cell cycle arrest at G1/S (33). p53 appears to be a downstream target of p19 (34,35) and p19 can inhibit the activity of Mdm2 by reducing the activity of p53, thus inducing cell growth arrest or increased apoptosis (36). Ezoe et al found that p16, p19 and p27 exhibit synergy in promoting stable hematopoietic stem cell function (37). We found that expression levels of p16 and p53 in Pthrp KI mouse bone were significantly higher than in wild-type mice and the increased expression of these genes in p27-/-Pthrp KI mouse bone was reduced but still elevated compared to wild-type mice, Consequently other cell cycle inhibitors such as p16, or p53, may compensate for the absence of p27 leading to only partial rescue of the skeletal dysplasia phenotype of the Pthrp KI mouse when p27 is deleted.
This article is protected by copyright. All rights reserved
14
We previously found that proliferation of BM-MSCs derived from Bmi-1-/- mice was significantly reduced, whereas apoptosis was significantly increased; furthermore, p16, p19 and p27 in BM-MSCs derived from Bmi-1-/- mice were significantly upregulated (38). As a consequence Bmi-1 through inhibition of p16, p19 and p27, maintained self-renewal capacity of BM-MSCs and promoted their differentiation into osteoblasts, while inhibiting their differentiation into adipocytes. In previous studies we found that the expression of Bmi-1 in Pthrp KI mouse bone tissue was significantly reduced and its nuclear translocation was significantly reduced (7). Our current results show that the expression of Bmi-1 in p27-/ -Pthrp KI mouse bone was partially increased, indicating that deletion of the PTHrP NLS and C- terminus may reduce Bmi-1 but reducing the expression levels of p27 partially prevented this reduction. Consequently p27 may function upstream of Bmi-1.
Overall therefore the results of this study and of our previous studies provide a model that suggests that the PTHrP NLS and C-terminus inhibit p27, followed by an increase in Bmi-1 to inhibit the expression of p16 and p53, thus enhancing the proliferation of chondrocyte and BM-MSCs, promoting BM-MSCs to differentiate into osteoblasts, and thus increasing skeletal growth and development (Fig. 7). This process may be limited by the capacity of Bmi-1 to reduce p27, thus preventing unregulated skeletogenesis.
In summary, this study not only clarifies mechanisms of the PTHrP NLS and C-terminus in promoting bone formation and development, but may also provide experimental and theoretical evidence for potential utilization of the PTHrP NLS and C-terminus as a drug target to promote bone formation and development.
This article is protected by copyright. All rights reserved
15
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81100624 to M.Z.), from the National Basic Research Program of China (2012CB966902 and 2014CB942900), from and the Basic Research Program of Chongqing (CSTC2013jcyjC00009) to D.M. and from the Canadian Institutes of Health Research (CIHR) to D.G. .
Author Contributions Conceived and designed the experiments: DM. Performed the experiments: MZ JZ ZD YZ RW. Analyzed the data: MZ JZ DM. Contributed reagents/materials/analysis tools: AK DG. Wrote the paper: MZ DM DG.
This article is protected by copyright. All rights reserved
16
References 1.
Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF 1987 Identification of a novel 17,000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 262(15):7151-6.
2.
McCauley LK, Martin TJ 2012 Twenty-five years of PTHrP progress: from cancer hormone to multifunctional cytokine. J Bone Miner Res 27(6):1231-9.
3.
Wysolmerski JJ, Stewart AF 1998 The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Annu Rev Physiol 60:431-60.
4.
Wu TL, Vasavada RC, Yang K, Massfelder T, Ganz M, Abbas SK, Care AD, Stewart AF 1996 Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 271(40):24371-81.
5.
Cornish J, Callon KE, Nicholson GC, Reid IR 1997 Parathyroid hormone-related protein-(107-139) inhibits bone resorption in vivo. Endocrinology 138(3):1299-304.
6.
Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15(8):4064-75.
7.
Miao D, Su H, He B, Gao J, Xia Q, Zhu M, Gu Z, Goltzman D, Karaplis AC 2008 Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein. Proc Natl Acad Sci U S A 105(51):20309-14.
8.
Toribio RE, Brown HA, Novince CM, Marlow B, Hernon K, Lanigan LG, Hildreth BE, 3rd, Werbeck JL, Shu ST, Lorch G, Carlton M, Foley J, Boyaka P, McCauley LK, Rosol TJ 2010 The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. FASEB J 24(6):1947-57.
9.
Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE 1997 Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev 11(11):1464-78.
10.
Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP 1996 Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382(6589):325-31.
11.
Hong YB, Kang HJ, Kim HJ, Rosen EM, Dakshanamurthy S, Rondanin R, Baruchello R, Grisolia G, Daniele S, Bae I 2009 Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells. Exp Mol Med 41(3):151-60.
12.
Zhang HY, Du ZX, Liu BQ, Gao YY, Meng X, Guan Y, Deng WW, Wang HQ 2009 Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin. Exp Mol Med 41(5):362-9.
13.
Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM 1996 A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85(5):733-44.
14.
Chien WM, Garrison K, Caufield E, Orthel J, Dill J, Fero ML 2007 Differential gene expression of p27Kip1 and Rb knockout pituitary tumors associated with altered growth and angiogenesis. Cell Cycle 6(6):750-7.
15.
Kim JA, Hong S, Lee B, Hong JW, Kwak JY, Cho S, Kim CC 2007 The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16INK4A-cyclin D1/cdk4 and p21waf1, p27kip1-cyclin E/cdk2 pathways. Cell Immunol 245(1):16-23.
16.
Drissi H, Hushka D, Aslam F, Nguyen Q, Buffone E, Koff A, van Wijnen A, Lian JB, Stein JL, Stein GS 1999 The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts. Cancer Res 59(15):3705-11.
17.
Fiaschi-Taesch N, Sicari BM, Ubriani K, Bigatel T, Takane KK, Cozar-Castellano I, Bisello A, Law B, Stewart AF 2006 Cellular mechanism through which parathyroid hormone-related protein induces proliferation in
This article is protected by copyright. All rights reserved
17
arterial smooth muscle cells: definition of an arterial smooth muscle PTHrP/p27kip1 pathway. Circ Res 99(9):933-42. 18.
Fiaschi-Taesch N, Sicari B, Ubriani K, Cozar-Castellano I, Takane KK, Stewart AF 2009 Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1. Endocrinology 150(3):1429-39.
19.
Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A 1996 Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 85(5):721-32.
20.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA 2007 Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917-20.
21.
Xue Y, Zhang Z, Karaplis AC, Hendy GN, Goltzman D, Miao D 2005 Exogenous PTH-related protein and PTH improve mineral and skeletal status in 25-hydroxyvitamin D-1alpha-hydroxylase and PTH double knockout mice. J Bone Miner Res 20(10):1766-77.
22.
Liu J, Lv F, Sun W, Tao C, Ding G, Karaplis A, Brown E, Goltzman D, Miao D 2011 The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH. PLoS Genet 7(9):e1002294.
23.
Miao D, Bai X, Panda D, McKee M, Karaplis A, Goltzman D 2001 Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition. Endocrinology 142(2):926-39.
24.
Samadfam R, Xia Q, Goltzman D 2007 Co-treatment of PTH with osteoprotegerin or alendronate increases its anabolic effect on the skeleton of oophorectomized mice. J Bone Miner Res 22(1):55-63.
25.
Samadfam R, Xia Q, Goltzman D 2007 Pretreatment with anticatabolic agents blunts but does not eliminate the skeletal anabolic response to parathyroid hormone in oophorectomized mice. Endocrinology 148(6):2778-87.
26.
Boras-Granic K, Dann P, Vanhouten J, Karaplis A, Wysolmerski J 2014 Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production. PLoS One 9(5):e90418.
27.
Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9(10):1149-63.
28.
Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY 1996 Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85(5):707-20.
29.
Hengst L, Dulic V, Slingerland JM, Lees E, Reed SI 1994 A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc Natl Acad Sci U S A 91(12):5291-5.
30.
Koff A, Ohtsuki M, Polyak K, Roberts JM, Massague J 1993 Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta. Science 260(5107):536-9.
31.
Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massague J 1994 Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78(1):59-66.
32.
MacLean HE, Guo J, Knight MC, Zhang P, Cobrinik D, Kronenberg HM 2004 The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes. J Clin Invest 113(9):1334-43.
33.
Serrano M, Hannon GJ, Beach D 1993 A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366(6456):704-7.
34.
Zhang Y, Xiong Y, Yarbrough WG 1998 ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92(6):725-34.
35.
Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ 1998 Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci U S A 95(14):8292-7.
This article is protected by copyright. All rights reserved
18
36.
Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, Cordon-Cardo C, DePinho RA 1998 The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 92(6):713-23.
37.
Ezoe S, Matsumura I, Satoh Y, Tanaka H, Kanakura Y 2004 Cell cycle regulation in hematopoietic stem/progenitor cells. Cell Cycle 3(3):314-8.
38.
Zhang HW, Ding J, Jin JL, Guo J, Liu JN, Karaplis A, Goltzman D, Miao D 2010 Defects in mesenchymal stem cell self-renewal and cell fate determination lead to an osteopenic phenotype in Bmi-1 null mice. J Bone Miner Res 25(3):640-52.
This article is protected by copyright. All rights reserved
19
Figure legends
Fig. 1 Expression profile of genes differentially expressed by microarray in bone tissue and the effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice (A) The differential expression of genes related to osteogenesis and cell cycle control between WT and Pthrp KI mouse bone tissue. (B) Confirmed results of microarray using real-time RT-PCR. (C) Survival curves of wild-type (WT), p27-/-, Pthrp KI and p27-/-Pthrp KI mice. (D) Body weight of 2-week-old mice. (E) Whole body view and (F) side view of 2-week-old mice. *：p
The p27 Pathway Modulates The Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone-Related Peptide† Min Zhu1*, Jing Zhang2*, Zhan Dong1, Ying Zhang1, Rong Wang1, Andrew Karaplis3, David Goltzman4, and Dengshun Miao1 1
State Key Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells, Department of Anatomy, Histology and Embryology, Nanjing Medical University, Nanjing 210029, Jiangsu, People's Republic of China; 2Department of Human Anatomy, Basic Medical College of Nanchang University, Nanchang 330046, Jiangxi, People's Republic of China; 3Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Canada; 4Calcium Research Laboratory, McGill University Health Centre and Department of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada * These authors contributed equally to this work. Corresponding author: Dengshun Miao, M.D., Ph.D. State Key Laboratory of Reproductive Medicine, The Research Center for Bone and Stem Cells Nanjing Medical University, Nanjing, Jiangsu, 210029, The People’s Republic of China Tel & FAX: 011-86-25-8686-2015 E-mail: [email protected]
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/jbmr.2544] Additional Supporting Information may be found in the online version of this article. Initial Date Submitted December 16, 2014; Date Revision Submitted April 15, 2015; Date Final Disposition Set April 23, 2015
Journal of Bone and Mineral Research This article is protected by copyright. All rights reserved DOI 10.1002/jbmr.2544
This article is protected by copyright. All rights reserved
1
Abstract Parathyroid hormone–related peptide (PTHrP) 1–84 knock-in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild type (WT) littermates. We found that the expression levels of p27, p16 and p53 were significantly up-regulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice which were homozygous for both p27 deletion and the Pthrp KI mutation (p27-/-Pthrp KI). We then compared p27-/-Pthrp KI mice with p27-/-, Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of PCNA positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and ALP-, type I collagen- and osteocalcin-positive bone areas were increased in p27-/- mice, and reduced in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however these parameters were increased in p27-/-Pthrp KI mice compared to Pthrp KI mice. As well, protein expression levels of PTHR, IGF-1 and Bmi-1, and the numbers of total CFU-f and ALP positive CFU-f were similarly increased in p27-/-Pthrp KI mice compared to Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the p27 pathway may function downstream in the action of PTHrP to regulate skeletal growth and development. This article is protected by copyright. All rights reserved Keywords：PTHrP, p27, skeletal growth, osteoblastic bone formation.
This article is protected by copyright. All rights reserved
2
Introduction Parathyroid hormone-related peptide (PTHrP), which was first identified in cancers associated with humoral hypercalcemia (1), is a pleiotropic factor with multiple physiological functions, including the regulation of morphogenesis, cell proliferation and differentiation, and transplacental calcium transport (2,3). PTHrP is a polyhormone which can be translated and processed into several smaller bioactive forms. The N-terminal region of PTHrP, i.e., PTHrP (1–36), mediates ‘‘classical’’ PTH-like effects. The midregion, i.e., PTHrP (37–86), has been implicated in placental calcium transport (4), and PTHrP (108–139), has been reported to inhibit bone resorption (5). A bipartite nuclear localization signal (NLS) is located in PTHrP (87–107) and can translocate heterologous plasma proteins into the nucleolus (6).
To examine the functional consequences of nuclear localization of PTHrP in vivo, we had previously generated a mouse expressing a partial ablation of Pthrp, that is, a Pthrp “knockin (KI) mouse”. Pthrp KI mice express PTHrP (1–84), a truncated form of PTHrP that is missing the NLS and the C-terminal region but can still signal through its cell surface receptor (7). Pthrp KI mice exhibit retarded growth, skeletal dysplasia and early senescence, leading to a rapid demise after birth (7). These results suggested that the NLS and C terminus of PTHrP play a critical role in bone development and function by stimulating the proliferation and differentiation of bone marrow cells. Other in vivo genetic mouse models have also indicated that the mid-region and C-terminus of PTHrP are functional (8).
Cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves
cyclin
dependent
kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different This article is protected by copyright. All rights reserved
3
phases
of the cell
cycle.
One CKI of importance is p27KIP1. The p27KIP1 protein was first identified
as an inhibitor of cyclin E/CDK2 complexes during TGFβ-induced G1 arrest (9). The p27KIP1 protein inhibits cyclin/CDK activity by binding cyclin/CDK complexes through its N-terminal domain, thus blocking ATP binding, and physically occluding the catalytic cleft of the CDK(10)，(11,12).
The p27KIP1 null (p27-/-) mouse shows an overall increase in cell proliferation, resulting in approximately 30% increase in body size and multiple organ hyperplasia (13-15). Bone marrow cells from p27 null mice exhibited increased proliferative activity and form an increased number and larger size of osteoblastic colonies, which can further differentiate to the stage of mineralization (16). PTHrP induces proliferation in arterial smooth muscle cells via a PTHrP/p27KIP1 pathway in which PTHrP-induces proteasomal degradation of p27 (17). In contrast, mutant PTHrP devoid of the NLS, markedly inhibits arterial smooth muscle cell cycle and neointima formation by up-regulation of p27KIP1 (18). However, it is unclear whether the NLS and C terminus of PTHrP regulate bone formation via a p27KIP1 pathway. In this work, we generated compound mutant mice which are homozygous for both p27 deletion and the PTHrP KI mutation (p27 -/- PTHrP KI), and compared their phenotypes with those of p27 -/-, PTHrP KI and WT littermates.
This article is protected by copyright. All rights reserved
4
Materials and Methods Derivation and genotyping of mice The p27-/- mice (C57BL/6J hybrid background) (13) used in this study were purchased from the Jackson Laboratory. The Pthrp KI mice (C57BL/6J hybrid background) used in this study were generated and characterized as we previously described (7). p27+/- mice and Pthrp KI+/- mice were fertile and were mated to produce offspring heterozygous at both loci, which were then mated to generate p27-/-Pthrp KI-/- pups. All animal experiments were carried out in compliance with, and approval by, the Institutional Animal Care and Use Committee. The genotype of p27-/- and Pthrp KI mice was confirmed as described previously (7,19).
Microarray analysis and real-time RT-PCR RNA was isolated from mouse bone tissue, excluding the growth plate, using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. Microarray analysis and time RT-PCR were performed as described previously (20,21) and as described in detail in the Supplementary Materials and Methods.
Iconographic and histopathological analyses Femurs or tibiae were removed and analyzed by radiography, micro-computed tomography, histology, histochemistry and immunohistochemistry as we described previously (21).
Western blot analysis Proteins were extracted from bone tissue and immunoblotting was carried out as we described previously (22) and as described in detail in the Supplementary Materials and Methods.
This article is protected by copyright. All rights reserved
5
CFU-f assay and cytochemical staining Bone marrow cells were flushed out from tibiae and femurs of 2-week-old mice and 106 bone marrow cells were cultured in 36-cm2 petri dishes for 10 to 18 days. At the end of the culture period, cells were stained with methyl blue or cytochemically for ALP. Following staining, the positive areas of colony-forming units, fibroblastic (CFU-f) and ALP positive CFU-f (CFU-fap) were measured using computer-assisted image analysis as we described previously (23).
Statistical analysis Data from image analysis are presented as mean ± s.e.m. Statistical comparisons were made using a two-way ANOVA, with P2 fold up- or down-regulated genes, p < 0.05) (Supplementary Table 1), in the bone tissue of E18.5 Pthrp KI mice compared with their WT littermates; 11 genes (listed in Table 1) were related to BM-MSC differentiation and cell cycle control. The most dramatically down-regulated genes in Pthrp KI mice included V-ATPase, osterix, cyclin D1, cyclin E, CDK2, ALP and Runx2. In contrast, the expression of 4 genes, p16, p53, p27 and PPARγ2 were significantly up-regulated. Fewer genes were found to be differentially expressed based on the q-values than on the p-values. The expression levels (log scale) of the genes related to BM-MSC differentiation and cell cycle control were reordered and displayed in a heat map, with the spectrum ranging from green (low level) to red (high level), as presented in Fig. 1A. The genes related to BM-MSC differentiation included osterix, ALP, Runx2 and PPARγ2. The genes related to cell cycle control included cyclin D1, cyclin E, CDK2, p16, p53 and p27. We validated the microarray analysis data using real-time PCR. This was carried out using gene-specific primers and the same RNA sources used for the microarray analysis. The results found by real-time PCR corroborated those from the microarray analysis, as shown in Fig.1B. The genes that were significantly different in Pthrp KI mice and WT mice showed similar alterations in expression with both measurement techniques.
Previous in vitro studies have implicated the PTHrP nuclear localization signal in inducing proliferation in arterial smooth muscle cells by modulating p27 (17). In view of our finding of upregulated p27 in skeletal tissue of Pthrp KI mice we next determined whether the cell cycle inhibitor p27Kip1 was involved in the regulation by PTHrP of skeletal growth and development in This article is protected by copyright. All rights reserved
7
vivo. We therefore generated compound mutant mice which are homozygous for both p27 deletion and the Pthrp KI mutation (p27-/-Pthrp KI), and compared them with p27-/-, Pthrp KI, and WT littermates.
The effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice To determine whether the deletion of p27 can rescue the growth retardation and premature senescence caused by Pthrp KI, we examined the effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice. Our results showed that p27-/- mice and WT mice survived for more than 2 years in contrast to the mean survival time of about 2 weeks in Pthrp KI mice; deletion of p27 in the Pthrp KI mice prolonged survival in the p27-/-Pthrp KI mice to about 3 weeks (Fig. 1C). The body weight and habitus were increased significantly in p27-/- mice, but were reduced in both Pthrp KI and p27-/-Pthrp KI mice compared to their WT littermates; however they were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 1D-F).
The effect of deletion of p27 on the skeletal growth of Pthrp KI mice To assess whether the deletion of p27 can correct skeletal growth retardation observed in the Pthrp KI, we examined the effect of deletion of p27 on the length of long bones and the proliferation of chondrocytes of Pthrp KI mice. We found that the length of long bones, the width of the proliferating zone of cartilage growth plates and the percentage of PCNA positive chondrocytes were clearly increased in p27-/- mice, but were reduced significantly in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; nevertheless these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 2A-G).
The effect of deletion of p27 on bone volume and osteoblastic bone formation of Pthrp KI mice This article is protected by copyright. All rights reserved
8
To determine whether the deletion of p27 can correct premature osteoporosis caused by Pthrp KI, we examined the effect of deletion of p27 on bone volume and osteoblastic indices in Pthrp KI mice. BMD (Figs. 2A and C), epiphyseal, trabecular and cortical bone volume (Figs. 3A-F), osteoblast numbers (Figs. 4A and B), and ALP (Figs. 4C and D), and type I collagen (Col I) (Figs. 4E and F) and osteocalcin (OCN) (Figs. 4G and H) positive areas, and the percentage of Bmi1 positive osteoblasts relative to total osteoblasts were increased significantly in p27-/- mice, but were reduced significantly in both Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice.
The effect of deletion of p27 on the expression of osteogenesis-regulating molecules of Pthrp KI mice To further investigate whether the effect of deletion of p27 on osteoblastic activity of Pthrp KI mice is associated with alterations of expression levels of osteogenesis regulating molecules, we examined the protein expression levels in bone tissue of IGF-1, the type I PTH/PTHrP receptor (PTHR), protooncogene Bmi-1 and cell cyclin-dependent kinase inhibitors p16 and p53. The results showed that protein expression levels of IGF-1, PTHR and Bmi-1 were up-regulated in p27-/- mice, but were down-regulated in both Pthrp KI and p27-/- Pthrp KI mice compared to wild-type mice, however they were up-regulated in p27-/-Pthrp KI mice relative to Pthrp KI mice (Figs. 5A-D). We have previously reported that protein expression of PTHR in situ correlates with alkaline phosphatase staining of osteoblasts and quantitation of osteoblastic perimeter per bone perimeter (Ob.Pm/B.Pm) as well as with serum levels of osteocalcin (24,25). Consequently the altered expression levels of PTHR likely reflected changes in the number of osteoblastic cells that were observed. In contrast, protein expression levels of p53 and p16 were up-regulated in all three mutant models ie p27-/-, Pthrp KI and p27-/-Pthrp KI mice compared to WT mice; however they were up-regulated This article is protected by copyright. All rights reserved
9
more in Pthrp KI mice compared to p27-/-Pthrp KI mice (Figs. 5A, E and F).
The effect of deletion of p27 on the proliferation of BM-MSCs and differentiation into osteoblasts of Pthrp KI mice To assess whether the effect of deletion of p27 on osteoblasts of Pthrp KI mice was associated with alterations of the recruitment and differentiation of BM-MSCs, CFU-f assays were performed and the alterations in expression of genes related to osteogenic cell differentiation were determined. We found that CFU-f and ALP positive area and the gene expression levels of Runx2, ALP, type I collagen, osteocalcin and Bmi1 were increased significantly in p27-/- mice compared to WT mice, but were decreased significantly in Pthrp KI mice compared to WT mice. However, these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 6A-I). We also examined the expression levels of mRNA encoding the N-terminal domain of PTHrP and encoding PTHR in the osteogenic cells resulting from 10 day BM-MSC cultures. Results revealed that mRNA expression level of the N-terminus of PTHrP (Pthlh) was increased significantly in p27-/- mice, but was not altered significantly in Pthrp KI and p27-/-Pthrp KI mice compared to WT mice (Fig. 6J). The mRNA expression level of PTHR was increased significantly in p27-/- mice and decreased significantly in Pthrp KI mice, however, it was not altered in p27-/-Pthrp KI mice compared to WT mice (Fig. 6K).
The effect of deletion of p27 on osteoclastic bone resorption of Pthrp KI mice To investigate whether the effect of deletion of p27 on the alteration of bone volume of Pthrp KI mice is associated with the alteration of osteoclastic bone resorption, we examined the effect of deletion of p27 on osteoclastic bone resorption of Pthrp KI mice. The results showed that the ratio of RANKL/OPG and the TRAP positive osteoclast surface were increased in p27-/- mice, but were This article is protected by copyright. All rights reserved
10
decreased significantly in Pthrp KI and p27-/-Pthrp KI mice compared to WT mice. However, these parameters were increased significantly in p27-/-Pthrp KI mice compared to Pthrp KI mice (Figs. 6K-M).
This article is protected by copyright. All rights reserved
11
Discussion In this study, we used a genetic approach to gain insight into the role of p27 in contributing to the skeletal phenotype of Pthrp KI mice and to determine whether p27 might function downstream of PTHrP NLS and C-terminus in modifying bone formation and development.
Pthrp KI mice showed growth retardation and muscle atrophy, hyperkeratosis, body fat reduction and poor bone development as well as other phenotypes (7). It has recently been reported that although Pthrp KI mice exhibit defects in mammary mesenchyme differentiation and mammary duct outgrowth, the mammary buds in PTHrP (1-84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function (26). To address this issue we examined expression of mRNA encoding N-terminal PTHrP in osteoblastic cells derived from BMSCs from Pthrp KI mice as well as from WT and the other mutants used in our studies and found that expression levels of mRNA encoding N-terminal PTHrP were not significantly decreased in Pthrp KI osteoblastic cells. Our results are consistent with our previous observations in embryonic fibroblasts, suggesting that the osteogenesis defects occurring in Pthrp KI mice resulted from the loss of PTHrP nuclear function (7).
Cyclin-dependent kinase inhibitors are an important class of negative regulators of cell cycle proteins, of which p27 is a key member. P27 functions primarily by binding to cyclin-dependent protein kinases or cyclin-CDK complexes, to achieve cyclin-dependent protein kinase activity inhibition, thereby negatively regulating the cell cycle (27). Previous studies have shown that p27 knockout mice showed accelerated growth, and multi-organ hypertrophy due to increased cell proliferation (13,28). We found, via our microarray analysis, that p27 is a direct skeletal target of This article is protected by copyright. All rights reserved
12
PTHrP in vivo and that deletion of p27 in Pthrp KI mice partially rescued the phenotype of the growth retardation of Pthrp KI mice. Osteoblastic cells differentiate in bone marrow from BM-MSCs, which are pluripotent stem cells having the potential to differentiate into a variety of cell lineages. Drissi et al found that the ability of BM-MSCs to progress along the osteoblastic lineage was significantly higher in BM-MSCs derived from p27-/ - mice compared to those from with WT mice (16). Our data confirmed the above results and also showed that increased osteoblastic differentiation of BM-MSCs occurs when p27 is deleted from Pthrp KI mice as compared to BM-MSCs derived from Pthrp KI mice. Consequently p27 appears to lie downstream of the PTHrP NLS and C-terminus in stimulating osteoblastic differentiation from BM-MSCs. Fiaschi-Taesch et al found that specific mutations of PTHrP NLS can increase the expression levels of p27, such that vascular smooth muscle cells undergo growth arrest in vitro (18). Consequently PTHrP inhibition of the expression of p27 can promote the proliferation of vascular smooth muscle (17). Our studies show that a similar mechanism may occur in vivo with respect to skeletal growth and development and osteoblastic bone formation. Nevertheless a limitation of our study is that the use of a global knockout of p27 and of a model of global ablation of the NLS and C-terminus of PTHrP does not preclude the fact that indirect regulation of PTHrP action may still occur in the p27-/-Pthrp KI mouse model we have used. Consequently targeted gene ablation in skeletal cells would be important to do in future studies.
In vivo, compared with 2-week-old Pthrp KI mice, the weight, long bone length, width of the growth plate, chondrocyte proliferation percentage, bone mineral density, bone volume, osteoblastic bone formation and osteoclastic bone resorption parameters
were all increased significantly in
p27-/-Pthrp KI mice. P27 was first identified as a CDK inhibitors due to its ability to block the activity of cyclin E/ CDK2 and cyclin A/ CDK2 in cells arrested in G1 by lovastatin, TGF-β and This article is protected by copyright. All rights reserved
13
contact inhibition (29-31). Our results demonstrated that mRNA expression levels of cyclin E and CDK2 were down-regulated significantly, whereas mRNA expression levels of p27 were up-regulated significantly in Pthrp KI mouse bone. These results suggest that deletion of p27 improved skeletal growth and development in Pthrp KI mice is, at least partially, mediated by up-regulating cyclin E and CDK2. We also found that the expression of Bmi-1 at both gene and protein levels and Bmi1 positive osteoblasts were increased in p27-/- mouse bone, but were decreased in Pthrp KI mouse bone and increased in Pthrp KI mouse bone by deletion of p27. However, it remains to be investigated how p27 regulates Bmi-1.
Nevertheless the deletion of p27 in Pthrp KI mice only modestly, although significantly, improved the Pthrp KI mouse skeletal dysplasia phenotype (32);
consequently it is likely that other
mechanisms, beside p27 are driving the Pthrp KI phenotype and that other genes lie downstream of the PTHrP NLS and C-terminus in modifying skeletal growth and development..Previous studies have shown that p16 can selectively inhibit the activities of cyclin D:CDK4/6 complexes, preventing phosphorylation by pRb, and causing cell cycle arrest at G1/S (33). p53 appears to be a downstream target of p19 (34,35) and p19 can inhibit the activity of Mdm2 by reducing the activity of p53, thus inducing cell growth arrest or increased apoptosis (36). Ezoe et al found that p16, p19 and p27 exhibit synergy in promoting stable hematopoietic stem cell function (37). We found that expression levels of p16 and p53 in Pthrp KI mouse bone were significantly higher than in wild-type mice and the increased expression of these genes in p27-/-Pthrp KI mouse bone was reduced but still elevated compared to wild-type mice, Consequently other cell cycle inhibitors such as p16, or p53, may compensate for the absence of p27 leading to only partial rescue of the skeletal dysplasia phenotype of the Pthrp KI mouse when p27 is deleted.
This article is protected by copyright. All rights reserved
14
We previously found that proliferation of BM-MSCs derived from Bmi-1-/- mice was significantly reduced, whereas apoptosis was significantly increased; furthermore, p16, p19 and p27 in BM-MSCs derived from Bmi-1-/- mice were significantly upregulated (38). As a consequence Bmi-1 through inhibition of p16, p19 and p27, maintained self-renewal capacity of BM-MSCs and promoted their differentiation into osteoblasts, while inhibiting their differentiation into adipocytes. In previous studies we found that the expression of Bmi-1 in Pthrp KI mouse bone tissue was significantly reduced and its nuclear translocation was significantly reduced (7). Our current results show that the expression of Bmi-1 in p27-/ -Pthrp KI mouse bone was partially increased, indicating that deletion of the PTHrP NLS and C- terminus may reduce Bmi-1 but reducing the expression levels of p27 partially prevented this reduction. Consequently p27 may function upstream of Bmi-1.
Overall therefore the results of this study and of our previous studies provide a model that suggests that the PTHrP NLS and C-terminus inhibit p27, followed by an increase in Bmi-1 to inhibit the expression of p16 and p53, thus enhancing the proliferation of chondrocyte and BM-MSCs, promoting BM-MSCs to differentiate into osteoblasts, and thus increasing skeletal growth and development (Fig. 7). This process may be limited by the capacity of Bmi-1 to reduce p27, thus preventing unregulated skeletogenesis.
In summary, this study not only clarifies mechanisms of the PTHrP NLS and C-terminus in promoting bone formation and development, but may also provide experimental and theoretical evidence for potential utilization of the PTHrP NLS and C-terminus as a drug target to promote bone formation and development.
This article is protected by copyright. All rights reserved
15
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81100624 to M.Z.), from the National Basic Research Program of China (2012CB966902 and 2014CB942900), from and the Basic Research Program of Chongqing (CSTC2013jcyjC00009) to D.M. and from the Canadian Institutes of Health Research (CIHR) to D.G. .
Author Contributions Conceived and designed the experiments: DM. Performed the experiments: MZ JZ ZD YZ RW. Analyzed the data: MZ JZ DM. Contributed reagents/materials/analysis tools: AK DG. Wrote the paper: MZ DM DG.
This article is protected by copyright. All rights reserved
16
References 1.
Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF 1987 Identification of a novel 17,000-dalton parathyroid hormone-like adenylate cyclase-stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 262(15):7151-6.
2.
McCauley LK, Martin TJ 2012 Twenty-five years of PTHrP progress: from cancer hormone to multifunctional cytokine. J Bone Miner Res 27(6):1231-9.
3.
Wysolmerski JJ, Stewart AF 1998 The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor. Annu Rev Physiol 60:431-60.
4.
Wu TL, Vasavada RC, Yang K, Massfelder T, Ganz M, Abbas SK, Care AD, Stewart AF 1996 Structural and physiologic characterization of the mid-region secretory species of parathyroid hormone-related protein. J Biol Chem 271(40):24371-81.
5.
Cornish J, Callon KE, Nicholson GC, Reid IR 1997 Parathyroid hormone-related protein-(107-139) inhibits bone resorption in vivo. Endocrinology 138(3):1299-304.
6.
Henderson JE, Amizuka N, Warshawsky H, Biasotto D, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar localization of parathyroid hormone-related peptide enhances survival of chondrocytes under conditions that promote apoptotic cell death. Mol Cell Biol 15(8):4064-75.
7.
Miao D, Su H, He B, Gao J, Xia Q, Zhu M, Gu Z, Goltzman D, Karaplis AC 2008 Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein. Proc Natl Acad Sci U S A 105(51):20309-14.
8.
Toribio RE, Brown HA, Novince CM, Marlow B, Hernon K, Lanigan LG, Hildreth BE, 3rd, Werbeck JL, Shu ST, Lorch G, Carlton M, Foley J, Boyaka P, McCauley LK, Rosol TJ 2010 The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. FASEB J 24(6):1947-57.
9.
Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE 1997 Cyclin E-CDK2 is a regulator of p27Kip1. Genes Dev 11(11):1464-78.
10.
Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP 1996 Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382(6589):325-31.
11.
Hong YB, Kang HJ, Kim HJ, Rosen EM, Dakshanamurthy S, Rondanin R, Baruchello R, Grisolia G, Daniele S, Bae I 2009 Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells. Exp Mol Med 41(3):151-60.
12.
Zhang HY, Du ZX, Liu BQ, Gao YY, Meng X, Guan Y, Deng WW, Wang HQ 2009 Tunicamycin enhances TRAIL-induced apoptosis by inhibition of cyclin D1 and the subsequent downregulation of survivin. Exp Mol Med 41(5):362-9.
13.
Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM 1996 A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85(5):733-44.
14.
Chien WM, Garrison K, Caufield E, Orthel J, Dill J, Fero ML 2007 Differential gene expression of p27Kip1 and Rb knockout pituitary tumors associated with altered growth and angiogenesis. Cell Cycle 6(6):750-7.
15.
Kim JA, Hong S, Lee B, Hong JW, Kwak JY, Cho S, Kim CC 2007 The inhibition of T-cells proliferation by mouse mesenchymal stem cells through the induction of p16INK4A-cyclin D1/cdk4 and p21waf1, p27kip1-cyclin E/cdk2 pathways. Cell Immunol 245(1):16-23.
16.
Drissi H, Hushka D, Aslam F, Nguyen Q, Buffone E, Koff A, van Wijnen A, Lian JB, Stein JL, Stein GS 1999 The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts. Cancer Res 59(15):3705-11.
17.
Fiaschi-Taesch N, Sicari BM, Ubriani K, Bigatel T, Takane KK, Cozar-Castellano I, Bisello A, Law B, Stewart AF 2006 Cellular mechanism through which parathyroid hormone-related protein induces proliferation in
This article is protected by copyright. All rights reserved
17
arterial smooth muscle cells: definition of an arterial smooth muscle PTHrP/p27kip1 pathway. Circ Res 99(9):933-42. 18.
Fiaschi-Taesch N, Sicari B, Ubriani K, Cozar-Castellano I, Takane KK, Stewart AF 2009 Mutant parathyroid hormone-related protein, devoid of the nuclear localization signal, markedly inhibits arterial smooth muscle cell cycle and neointima formation by coordinate up-regulation of p15Ink4b and p27kip1. Endocrinology 150(3):1429-39.
19.
Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A 1996 Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 85(5):721-32.
20.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA 2007 Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917-20.
21.
Xue Y, Zhang Z, Karaplis AC, Hendy GN, Goltzman D, Miao D 2005 Exogenous PTH-related protein and PTH improve mineral and skeletal status in 25-hydroxyvitamin D-1alpha-hydroxylase and PTH double knockout mice. J Bone Miner Res 20(10):1766-77.
22.
Liu J, Lv F, Sun W, Tao C, Ding G, Karaplis A, Brown E, Goltzman D, Miao D 2011 The abnormal phenotypes of cartilage and bone in calcium-sensing receptor deficient mice are dependent on the actions of calcium, phosphorus, and PTH. PLoS Genet 7(9):e1002294.
23.
Miao D, Bai X, Panda D, McKee M, Karaplis A, Goltzman D 2001 Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition. Endocrinology 142(2):926-39.
24.
Samadfam R, Xia Q, Goltzman D 2007 Co-treatment of PTH with osteoprotegerin or alendronate increases its anabolic effect on the skeleton of oophorectomized mice. J Bone Miner Res 22(1):55-63.
25.
Samadfam R, Xia Q, Goltzman D 2007 Pretreatment with anticatabolic agents blunts but does not eliminate the skeletal anabolic response to parathyroid hormone in oophorectomized mice. Endocrinology 148(6):2778-87.
26.
Boras-Granic K, Dann P, Vanhouten J, Karaplis A, Wysolmerski J 2014 Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production. PLoS One 9(5):e90418.
27.
Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9(10):1149-63.
28.
Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY 1996 Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85(5):707-20.
29.
Hengst L, Dulic V, Slingerland JM, Lees E, Reed SI 1994 A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc Natl Acad Sci U S A 91(12):5291-5.
30.
Koff A, Ohtsuki M, Polyak K, Roberts JM, Massague J 1993 Negative regulation of G1 in mammalian cells: inhibition of cyclin E-dependent kinase by TGF-beta. Science 260(5107):536-9.
31.
Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massague J 1994 Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78(1):59-66.
32.
MacLean HE, Guo J, Knight MC, Zhang P, Cobrinik D, Kronenberg HM 2004 The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes. J Clin Invest 113(9):1334-43.
33.
Serrano M, Hannon GJ, Beach D 1993 A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366(6456):704-7.
34.
Zhang Y, Xiong Y, Yarbrough WG 1998 ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92(6):725-34.
35.
Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ 1998 Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci U S A 95(14):8292-7.
This article is protected by copyright. All rights reserved
18
36.
Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L, Potes J, Chen K, Orlow I, Lee HW, Cordon-Cardo C, DePinho RA 1998 The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 92(6):713-23.
37.
Ezoe S, Matsumura I, Satoh Y, Tanaka H, Kanakura Y 2004 Cell cycle regulation in hematopoietic stem/progenitor cells. Cell Cycle 3(3):314-8.
38.
Zhang HW, Ding J, Jin JL, Guo J, Liu JN, Karaplis A, Goltzman D, Miao D 2010 Defects in mesenchymal stem cell self-renewal and cell fate determination lead to an osteopenic phenotype in Bmi-1 null mice. J Bone Miner Res 25(3):640-52.
This article is protected by copyright. All rights reserved
19
Figure legends
Fig. 1 Expression profile of genes differentially expressed by microarray in bone tissue and the effect of deletion of p27 on lifespan, body weight and habitus of Pthrp KI mice (A) The differential expression of genes related to osteogenesis and cell cycle control between WT and Pthrp KI mouse bone tissue. (B) Confirmed results of microarray using real-time RT-PCR. (C) Survival curves of wild-type (WT), p27-/-, Pthrp KI and p27-/-Pthrp KI mice. (D) Body weight of 2-week-old mice. (E) Whole body view and (F) side view of 2-week-old mice. *：p
