Insulin-Like Growth Factor-Independent Effects of Growth Hormone on Growth Plate Chondrogenesis and Longitudinal Bone Growth.

ORIGINAL

RESEARCH

INSULIN-LIKE GROWTH FACTOR INDEPENDENT EFFECTS OF GROWTH HORMONE ON GROWTH PLATE CHONDROGENESIS AND LONGITUDINAL BONE GROWTH Shufang Wu1,2, Wei Yang2, Francesco De Luca1 1

Section of Endocrinology and Diabetes, St. Christopher’s Hospital for Children, Drexel University College of Medicine, Philadelphia, PA.2Center for Translational Medicine, the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, 277 W Yanta Rd, Xi’an, Shaanxi710061. P.R.China

Growth Hormone (GH) stimulates growth plate chondrogenesis and longitudinal bone growth directly at the growth plate. However, it is not clear yet whether these effects are entirely mediated by the local expression and action of Insulin-like Growth Factor-1 (IGF-1) and Insulin-like Growth Factor-2 (IGF-2). To determine whether GH has any IGF-independent growth-promoting effects, we generated TamCart Igf1rflox/flox mice. The systemic injection of tamoxifen in these mice postnatally resulted in the excision of the Igf1 receptor (Igf1r) gene exclusively in the growth plate. TamCartIgf1rflox/flox tamoxifen-treated mice (KO mice) and their Igf1rflox/flox control littermates (C mice) were injected for 4 weeks with GH. At the end of the 4-week period, tibial growth and growth plate height of GH-treated KO mice were greater than those of untreated C or untreated KO. The systemic injection of GH increased the phosphorylation of JAK2 and STAT5B in the tibial growth plate of the C and KO mice. In addition, GH increased the mRNA expression of BMP-2 and the mRNA expression and protein phosphorylation of NF-␬B p65 in both C and KO mice. In cultured chondrocytes transfected with Igf1r siRNA, the addition of GH in the culture medium significantly induced thymidine incorporation and collagen X mRNA expression. In conclusion, our findings demonstrate that GH can promote growth plate chondrogenesis and longitudinal bone growth directly at the growth plate even when the local effects of IGF-1 and IGF-2 are prevented. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.

G

rowth Hormone (GH) and Insulin-like Growth factor 1 (IGF-1) are the most important hormones controlling longitudinal bone growth; the interaction between GH and IGF-1 in the regulation of statural growth has been studied for many years (1, 2). According to the original somatomedin hypothesis, GH induces bone growth by stimulating the production in the liver of IGF-1 (previously known as somatomedin) which, in turn, stimulates longitudinal bone growth at the growth plate (3– 8). This evidence, along with the detection of IGF-1 and IGF-2 expression not only in the liver but also in other tissues,

supported a revised somatomedin hypothesis, according to which GH modulates longitudinal bone growth via IGF-1 expressed in extrahepatic tissues (9 –12). Furthermore, the somatomedin hypothesis was subsequently challenged by studies demonstrating a direct growth-promoting effect of GH when injected in a rat tibial growth plate, with this effect limited only to the injected bone (13, 14). Further studies confirmed a direct effect of GH on longitudinal bone growth, proposing that GH acts by inducing the expression and the action of IGF-1 locally in the growth plate (1, 15–16).

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received December 8, 2014. Accepted April 17, 2015.

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doi: 10.1210/en.2014-1983

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IGF-independent effects of GH on bone growth

More recently, a transgenic mouse was developed in which the Igf1 gene was selectively deleted in the liver: this mouse exhibited significantly reduced circulating IGF-1 levels and dramatically increased GH levels. Interestingly, this animal’s body growth and femoral growth were unaffected compared to those of wild-type animals. In another study, targeted disruption of IGF-1 expression in mouse chondrocytes led to reduced bone length (17). These findings suggested that IGF-1 expressed in the growth plate could be the primary mediator of the GHdependent effects on longitudinal bone growth (18, 19). On the other hand, further evidence suggests that GH may have IGF-1-independent effects on bone growth. The concept that GH has direct, non-IGF-1dependent effects on the growth plate has been supported by studies showing that the growth plate germinal zone is expanded in Igf1 null mice (20). Given the absence of IGF-1 expression in these mice, high circulating GH levels may be responsible for the expanded germinal zone. Indirect evidence of an IGF-1-independent effect of GH on bone growth also results from two studies. In one study, it was demonstrated that tibial growth rate is reduced more in Ghr null mice than in Igf1 null mice between postnatal days 20 and 40, a time of peak GH effect on longitudinal bone growth (21). In another study, the authors demonstrated that the body size of mutant mice lacking both Ghr and Igf1 was smaller than that of mice lacking Igf1 or Ghr (22). However, in the Igf1 null mice IGF-2 expression in the growth plate is significantly increased (21); thus, it is possible that the IGF-1-independent effects of GH on growth plate chondrogenesis and longitudinal bone growth may be mediated by IGF-2. To determine whether GH can affect growth plate chondrogenesis and longitudinal bone growth without the mediation of IGF-1 or IGF-2, we used a transgenic mouse in which the type 1 Insulin-like Growth Factor Receptor (Igf1r) gene was conditionally ablated postnatally by Crelox recombination only in chondrocytes. Specifically, we made a tamoxifen (Tam)-inducible TamCartIgf1r-/- mouse that enabled us to conditionally ablate the Igf1r gene in chondrocytes during postnatal life after systemically injecting tamoxifen. In addition, to study the direct and specific effects of GH on growth plate chondrocyte function we used cultured chondrocytes transfected with siRNAs specific for Igf1r and/or Ghr.

Materials and Methods Generation of mice with cartilage-specific Igf1rdisruption Briefly, the inducible TamCartIgf1rflox/flox mice were generated by breeding Igf1rflox/flox mice (that carry loxP sequences flanking

Endocrinology

exon 3 of the gene) (23) (strain name: B6; 129-Igf1rtm2arge/J. These mice were purchased from Jackson Laboratories; stock number, 012 251) with mice expressing a CreERCart transgene, which encodes a fusion protein of the Cre recombinase and a mutated estrogen-responsive element to confer sensitivity to tamoxifen (24, 25) and is regulated by the ␣1 (II) promoter for cartilage-specific expression (24) (these mice were kindly donated by Dr. Susan Mackem). The resulting TamCartIgf1rflox/flox mice were viable and fertile in the absence of tamoxifen. To produce Igf1r gene knockout in the growth plate of TamCart Igf1r-/- mice during postnatal growth, TamCartIgf1rflox/ flox mice were injected with four doses of tamoxifen (0.2 mg/ mouse) at 2-day intervals beginning on day 5 after birth. From now on, we will refer to the TamCartIgf1r-/- mice as KO mice and to the Igf1rflox/flox control littermates as C mice.

Genotyping and determination of tissue-specific deletion of the Igf1r gene Genotyping was performed after extracting genomic DNA from tail snips and other tissues (heart, liver, spleen, lung, kidney, and the tibial growth plate) using DNeasy Blood and Tissue Kit (QIAGEN Inc., Valencia CA). Polymerase chain reaction (PCR) analyses of the DNA were performed to detect Cre and floxedIgf1r alleles using corresponding primer sets with standard conditions (30 seconds at 94°C, 30 seconds at 58°C, and 30 second at 72°C for 35 cycles). PCR was performed with a mixture of three primers (two forward primers, 5⬘-CTT CCC AGC TTG CTA CTC TAG G-3⬘ and 5⬘-TGA GAC GTA GCG AGA TTG CTG TA-3⬘, and a reverse primer, 5⬘-CAG GCTTGC AAT GAG ACA TGG G-3⬘) to detect 320-and 120-bp products from the excised and nonexcised gene alleles, respectively (26). The cre transgene was detected by PCR using the primers 5⬘-GAA AAT GCT TCT GTC CGT TTG C –3⬘ (forward) and 5⬘-ATT GCT GTC ACT TGG TCG TGG C –3⬘ (reverse) to amplify a 207-bp DNA product.

Animal care Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication (NIH) 85– 23, revised 1988]. All procedures were approved by the Institutional Animal Care and Use Committee of Drexel University College of Medicine. Male and female mice were maintained in a temperature(22°C), humidity-, and light-(12 hours of light, 12 hours of darkness)-controlled environment. Three-week old KO and C mice were housed separately and allowed ad libitum access to chow and water. At the beginning of the 4-weeks study period, mice were assigned to 6 groups, each one including 4 –7 mice: two untreated groups (C-untreated and KO-untreated), two groups treated with daily injections of recombinant mouse IGF-1 (5 mg/kg. PEPROTECH, Rocky Hill, NJ, Cat# 100 –11) throughout the study period (C-IGF-1-treated and KO-IGF-1-treated), and two groups treated daily with recombinant mouse GH (5 mg/kg. National Hormone & Peptide Program, Harbor-UCLA Medical Center, Torrance, CA) (C-GH-treated and KO-GH-treated). Weight and whole body length (from nose to anus) were measured weekly during the 4-week study period after the animals were sedated with an injection of ketamine hydrochloride (0.02 mg/100 g body weight) (Phoenix Pharmaceutical, Inc.) and


doi: 10.1210/en.2014-1983

acepromazine (0.1 mg/100 g body weight) (Roche Applied Science). Serial radiographs of the tibiae were obtained weekly, with each animal placed prone on an x-ray cassette. The lengths of both tibiae were measured on the radiograph, and the average value was calculated. At the end of the 4-week study period, blood was collected by ventricular puncture from each animal before euthanasia. Serum samples were obtained and stored at –20°C for GH and IGF-1 analysis. After euthanasia, tibial growth plates were removed; half of them were stored at – 80°C for subsequent RNA extraction and half were fixed with 4% paraformaldehyde overnight, decalcified with Decalcifier II (Surgipath, Richmond, IL) for 1 hour, and paraffin-embedded.

Measurement of serum hormones and metabolites After collection, serum was stored at – 80°C before analysis. GH levels were determined in duplicate by ELISA (Millipore Corporation, Billerica, MA) with intra- and interassay C.V of 1.7 to 4.3% and 3.2 to 4.9% respectively. IGF-1 levels were determined in duplicate by a mouse- specific Quantikine ELISA including a 500-fold dilution of all samples (R&D Systems, Inc. Minneapolis, MN) with intra- and interassay C.V. of 4.1 to 5.6% and 4.3 to 9.0% respectively.

Quantitative Histology At the end of the 4-week study period, we harvested 4 –7 tibiae for each treatment group. Three 5–7-␮m thick longitudinal sections from each bone were obtained and stained with hematoxylin/phloxine/saffron. Tibial growth plate epiphyseal, proliferative, and hypertrophic zone heights (␮m) were measured. All measurements were performed by a single observer blinded to the treatment regimen.The representative pictures shown in the figures were obtained at 20X magnification.

Immunohistochemistry To verify the ablation of IGF-1R in the tibial growth plate, we isolated tibiae from C and KO mice at DOL 14 (3 days after the last tamoxifen injection) and at the end of the 4-week treatment period. Three 5–7-␮m thick longitudinal sections were obtained from each growth plate, deparaffined in xylene and rehydrated in graded ethanol. Sections were incubated in 1% H2O2 for 10 minutes followed by three rinses with PBS. For digestion, 0.1% trypsin for 12 minutes was used at room temperature (RT) followed by a triple wash in PBS. After preincubation with 1.5% blocking serum for 30 minutes at RT, sections were incubated for 30 minutes at RT with rabbit polyclonal antibody raised against IGF-1R (1:150) (Cat. # SC-713, Santa Cruz Biotechnology Inc., Santa Cruz, CA). The secondary antibody was an antirabbit antibody conjugated with biotin, applied for 30 minutes at a dilution of 1:200. This step was followed by an incubation for 30 minutes with avidin and biotinylated horseradish peroxidase. The sections were then visualized with peroxide substrate for 5 minutes and mounted with Permount medium. Control experiments were performed using normal rabbit serum instead of the primary antibody.

Chondrocyte culture The cartilaginious portions of metatarsal bones isolated from C57BL/6N mouse embryos (dpc 20) were dissected, rinsed in PBS, then incubated in 0.2% trypsin for 1 hour and 0.2% col-


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lagenase for 3 hours. Cell suspension was aspirated repeatedly and filtered through a 70-␮m cell strainer, rinsed first in PBS then in serum-free DMEM, and counted. Chondrocytes were seeded in 100-mm dishes at a density of 5 ⫻ 104/cm2 in DMEM with 100U/ml penicillin and 100 ␮g/ mlstreptomycin, 50 ␮g/ ml ascorbic acid, and 10% FBS. The culture medium was changed at 72h intervals. Upon confluence, cells were subcultured and their chondrogenic phenotype confirmed by studying the expression of type-I collagen, type-II collagen and type X collagen by immunocytochemistry. In our culture conditions, the percentage of cells expressing type-I, type-II and type-X collagen was 3.9%, 97.1% and 22.0% respectively. Subsets of cells with confirmed chondrogenic phenotype (with at least 95% of the cells positive for type-II-collagen positive and negative for type-I-collagen negative) were treated for 24 hours without or with recombinant mouse GH (10ng/ml) or recombinant mouse IGF-1 (100 ng/ml).

siRNA transfection Chondrocytes were transfected with pools of siRNAs targeted for GH receptor (GHR) (sc-40 016, Santa Cruz), and IGF-1 receptor (IGF-1R) (sc-37 194, Santa Cruz), A pool of siRNAs consisting of a scrambled sequences was similarly transfected as control siRNA (Santa Cruz) and introduced into cells using LipofectAMINE 2000 (Invitrogen), according to the procedure recommended by the manufacturer. One day before transfection, cells were plated in 500 ␮l of growth medium without antibiotics, such that they were 30%–50% confluent at the time of transfection. The transfected cells were cultured in DMEM containing 10% FCS for 72 hours after transfection. 3

H-thymidine incorporation

To assess cell proliferation, we measured 3H-thymidine incorporation into newly synthesized DNA. At the indicated time points during the culture period, 2.5 ␮Ci/well of 3H-thymidine (25 Ci/mmol; Amersham, Piscataway, NJ) was added to the culture medium for an additional 3 hours. Cells were released by trypsin and collected onto glass fiber filters. Incorporation of 3 H-thymidine was measured by liquid scintillation counting. The data represent percentage of control from three independent experiments.

RNA extraction and Real time PCR Total RNA was extracted from the tibial growth plate and from cultured chondrocytes isolated from fetal (dpc20) mouse metatarsal growth plates using the QIAGEN RNeasy Mini kit (QIAGEN Inc., Valencia CA). The recovered RNA was further processed using first Strand cDNA Synthesis kit for RT-PCR (AMV)(Roche Diagnostics Corp. Indianapolis, IN) to produce cDNA. 1 ␮g of total RNA and 1.6 ␮g of oligo-p(dT)15 primer were incubated for 10 minutes at 25°C, followed by incubation for 60 minutes at 42°C in the presence of 20 Units AMV Reverse transcriptase and 50 Units of RNase inhibitor in a total 20␮l reaction. The cDNA products were directly used for PCR or stored at – 80°C for later analysis. Real-time quantitative PCR was carried out using StepOne Real time PCR System (Applied Biosystems, Foster City, CA) in a final volume of 25 ␮l containing 1 ␮l cDNA, 12.5 ␮l 2 x SYBR Green master mix (Applied Biosystems), 0.1 ␮M primers (Applied Biosystems) in DNAsefree water. Primers used were: mouse ␤-actin (forward 5⬘-TGT GAT GGT GGG AAT GGG TCA GAA-3⬘, reverse 5⬘-TGT GGT


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GCC AGA TCT TCT CCA TGT-3⬘); mouse IGF-1 (forward 5⬘-GGG CAT TGT GGA TGA GTG TTG CTT-3⬘, reverse 5⬘TGG AAC GAG CTG ACT TTG TAG GCT-3⬘); mouse IGF-2 (forward 5⬘-AAG AGT TCA GAG AGG CCA AAC-3⬘, reverse 5⬘-ACT GAT GGT TGC TGG ACA TC-3⬘); mouse GHR (forward 5⬘-CCT CCA TTA CCC AGA CAG TAG A-3⬘; reverse 5⬘-TTG CCA GTG ATG TGG GAT TAC-3⬘); mouse IGF-1R (forward 5⬘-GGA GGA GTT CGA GAC AGA GTA-3⬘; reverse 5⬘-CGA TGC GGT ACA GAG TGA AA-3⬘); mouse Collagen X (forward 5⬘-ATA AGA ACG GCA CGC CTA CGA TGT-3⬘, reverse 5⬘- CTG CAT TGG GCA ATT GGA GCC ATA-3⬘) and mouse BMP-2 (forward 5⬘-TTC TGT CCC TAC TGA TGA GTT TCT C-3⬘; reverse 5⬘-AAG TCA CTA GCA GTG GTC TTA CCT G-3⬘).The PCR conditions were: 50°C for 2 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Values were quantified using the comparative cycle threshold (CT) method (27), and samples were normalized to ß-actin.

Western blot Chondrocytes were harvested and immunoblotting was performed using equal amounts of protein (50 –100 ␮g) and the following primary antibodies: anti-␤actin (Cat. No.A-2066; Sigma), p-NF-␬B p65 (SC-33 039, Santa Cruz), rabbit polyclonal antibody against NF-␬B p65 (SC-372,Santa Cruz), p-Jak2 (Cat#9356, Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal antibody against Jak2 (SC-294, Santa Cruz), rabbit polyclonal antibody against p-STAT5 (Cat#4322, Cell Signaling Technology Inc.),rabbitpolyclonal antibody against STAT5 (SC-835,SantaCruz), goat polyclonal antibody against BMP-2 (SC-6895, Santa Cruz). The blots were developed using a horseradish-peroxidase-conjugated polyclonal donkey-anti rabbit IgG or bovine-anti goat IgG antibody and enhanced chemiluminescence system (GE healthcare).

Endocrinology

Serum levels of GH and IGF-1 in KO and C mice, untreated or treated with GH or IGF-1 Serum levels of IGF-1 were similar in untreated KO and C mice. IGF-1 levels were higher in mice treated with IGF-1 or GH when compared to untreated mice, both in the KO and C groups (figure 2A). With respect to serum GH levels, they were higher in KO or C mice treated with GH compared to GH levels in untreated mice (figure 2B). Effects of systemic IGF-1 and GH on body growth, tibial growth, and tibial growth plate morphology of KO and C mice At the beginning of the 4-week study, the mean body length and tibial length of the KO and C mice were equivalent (data not shown). By the end of the 4-week treatment period, body and tibial growth, and the height of the whole growth plate, of untreated KO mice were significantly smaller than those of untreated C mice (figures 3A-B; Supplemental figure 2). In addition, body growth and tibial growth of C mice treated with GH or IGF-1 were significantly greater than those of untreated C mice (figure 3AB). In KO mice, body and tibial growth of IGF-1-treated

Statistics All data are expressed as the mean ⫹/- SE. Data were analyzed using SPSS version 13.0 (SPSS Inc, Chicago, IL) and statistical significance was determined by one-way ANOVA followed by Tukey-Kramer’s multiple comparison post hoc test. P values less than 0.05 were considered statistically significant.

Results Tissue-specific deletion of the Igf1r gene We confirmed the specificity of gene excision in the KO mice by PCR analyses of gDNA from different tissues. As shown in figure 1A, gene excision occurred only in the growth plate from the KO mice but not in other tissues from the same animals or in the growth plate of C mice. Ablation of Igf1r gene expression in the tamoxifen-treated KO mice was confirmed by a decrease (by ⬎ 90%) in its mRNA expression by qPCR (figure 1B) and in protein expression by immunohistochemistry (Supplemental figure 1).

Figure 1. Ablation of the Igf1r gene in TamCartIgf1r-/-(KO) mice. A:PCR analyses of gDNA extracted from different tissues of the TamCart Igf1r-/-(KO) and of the Igf1rflox/flox littermates (C) with primer sets for the Cre transgene, floxed-Igf1r allele, and sequences after gene excision (Igf1r) as described in “Materials and Methods.”B: At the end of 4-week study period, total RNA was extracted from the liver and growth plate of KO and C mice (n ⫽ 4 –7/group) and expression of Igf1r mRNA was detected by real-time PCR. The mRNA level was normalized by ␤-actin in the same samples.


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mice was similar to that of untreated mice, and significantly smaller than that of IGF-1-treated C mice. Of note, body and tibial growth of GH-treated KO mice were greater than those of untreated C or untreated KO mice; they were also greater than those of IGF-1-treated KO mice (figure 3A-B). The reduced growth plate height in untreated KO mice was due to a significantly reduced height of epiphyseal, proliferative and hypertrophic zones (figure 4A-D; Supplemental figure 2). In addition, the height of the growth plate of C mice treated with GH or IGF-1 were significantly greater than those in untreated C mice (figure 4A; Supplemental figure 2). In KO mice, the growth plate height of IGF-1-treated mice was similar to that of untreated mice, and significantly smaller than that of IGF-1-treated C mice. In contrast, the growth plate height of GH-treated KO mice was greater than that of untreated C or untreated KO mice; it was also greater than that of IGF-1-treated KO mice (figure 4A; Supplemental figure 2). With respect to the effects on the different zones of the growth plate, IGF-1 significantly increased the

Figure 2. Serum levels of IGF-1and GH in C and KO mice treated with or without IGF-1 or GH. Three-week old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of 4-weeks study period, serum IGF-1 (A) and GH (B) levels were measured by ELISA in C and KO mice. Results are expressed as mean⫹/- S.E


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height of the epiphyseal, proliferative, and hypertrophic zones in the C mice, but not in the KO mice (figure 4B-D; Supplemental figure 2). GH increased the height of all three zones of the growth plate, both in the C and in the KO mice (figure 4B-D; Supplemental figure 2). Effects of systemic GH and IGF-1 on the mRNA expression of Igf1, Igf2, Ghr, and Igf1r in the tibial growth plate of KO and C mice In C mice, both IGF-1 and GH induced the expression of Igf1 and Igf2 in the growth plate (figure 5) compared to untreated mice. In addition, the injection of IGF-1 and GH induced the expression of Igf1r and Ghr respectively. In KO mice, IGF-1 injection induced the mRNA expression of Igf1 and Igf2, but did not modify the expression of either the Igf1r or Ghr. Lastly, GH stimulated the expression of both Igfs and Ghr, but not that of Igf1r (figure 5).

Figure 3. Effects of systemic GH and IGF-1 on body growth and tibial growth of KO and C mice. Three-week old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. Whole body length (from nose to anus) and tibial length (average length of both tibiae measured on radiographs) were obtained weekly. The difference in body length and tibial length (in cm) measured at the beginning and at the end of the 4-week study period represents the whole body growth (panel A) and tibial growth (panel B).


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Effects of systemic GH and IGF-1 on Ghrdependent and Igf1r-dependent signaling pathways in the tibial growth plate of KO and C mice The systemic injection of GH increased the phosphorylation of JAK2 and STAT5B, (but not of Akt) in the tibial growth plate, both in the C and KO mice, compared to untreated mice (figure 6A-B; Supplemental figure 3). IGF-1 induced the phosphorylation of Akt in C mice but not in KO mice. These findings confirm the lack of activation of the main Igf1r-dependent (and Ir-dependent) signaling pathway in the growth plate of KO mice treated with IGF-1 or GH (figure 6C; Supplemental figure 3). Effects of systemic GH on BMP-2 mRNA expression and NF-␬B p65 mRNA expression and protein phosphorylation in the tibial growth plate of KO and C mice To determine whether the IGF-independent effects of GH on bone growth and growth plate chondrogenesis are mediated by BMP-2 and/or NF-␬B p65, we evaluated the expression and activation of these two transcription factors. Compared to untreated mice, the systemic injection of GH increased the mRNA expression of BMP-2 and the mRNA expression and protein phosphorylation of NF-␬B p65 in the tibial growth plate, both in the C and KO mice

Endocrinology

(7A-B; Supplemental figure 3). The systemic injection of IGF-1 induced the mRNA expression of BMP-2 and the mRNA expression and protein phosphorylation of NF-␬B p65 in the growth plate of C mice, but had no effect in the KO mice (figure 7A-B; Supplemental figure 3). Effects of GH and IGF-1 in cultured chondrocytes transfected with Ghr siRNA and/or Igf1r siRNA To validate the specificity of the siRNA-mediated inhibition of the target gene expression, we analyzed the mRNA expression of the target genes in transfected chondrocytes by real-time PCR. In control siRNA-transfected chondrocytes, the mRNA expression of Ghr, Igf1r, or Ir was unchanged when compared to that detected in untransfected cells (Supplemental figure 4). Cells transfected with Ghr siRNA or Igf1r siRNA exhibited ⬃ 80% reduction of Ghr and Igf1rmRNA expression respectively; Ir mRNA expression was not modified by the transfection of either Igf1r siRNA or Ghr siRNA (Supplemental figure 4). In control siRNA-transfected chondrocytes, the addition of GH and IGF-1 in the culture media significantly induced thymidine incorporation (figure 8A) and collagen X mRNA expression (figure 8B), compared to untreated control siRNA-transfected cells. In Igf1r siRNA-transfected chondrocytes, IGF-1 did not modify either thymidine incorporation or collagen X mRNA expression; in

Figure 4. Effects of systemic GH and IGF-1 on the tibial growth plate histology of KO and C mice. Three–week old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of the 4-week study period, 4 –7 tibiae for each treatment group were isolated. Three 5–7 ␮m thick longitudinal sections were obtained from each bone and stained with hematoxylin/ phloxine/saffron. The growth plate zone heights were measured by one observer blinded to the treatment groups. A: Whole growth plate. B: Epiphyseal zone. C: Proliferative zone. D: Hypertrophic zone. Results are expressed as mean⫹/- S.E.


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contrast, GH increased both. Similarly, in Ghr siRNAtransfected chondrocytes, GH had no effects on thymidine incorporation or on collagen X mRNA expression, while both parameters were significantly increased by IGF-1. Lastly, the cotransfection of chondrocytes with Igf1r siRNA and Ghr siRNA prevented any effects of IGF-1 and GH on thymidine incorporation and collagen X mRNA expression.

Figure 5. Effects of systemic GH and IGF-1 on the mRNA expression of Igf1, Igf2, Igf1r, and Ghr in the tibial growth plate of KO and C mice. Three-week old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of the treatment period, total RNA was extracted from the tibial growth plate of KO and C mice. mRNA expression was quantitated by real-time PCR. The relative expression levels of mRNA were normalized by ␤-actin in the same samples. Results were expressed as fold change compared to untreated control (mean ⫾S.E).


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Discussion Our findings indicate that the postnatal ablation of Igf1r in the mouse growth plate results in diminished body and tibial growth. Despite the lack of Igf1r expression in the growth plate, postnatal systemic administration of GH induces body and tibial growth, as well as cartilage formation in the growth plate. As a result, these findings indicate that GH may promote longitudinal bone growth through IGF-independent mechanisms. It has long been debated whether GH modulates statural growth exclusively by inducing IGF-1 synthesis and action, systemically and/or locally within the growth plate. Igf1 null mice exhibit severe retardation of body growth at birth, and their postnatal growth is also significantly impaired (28 –30). Furthermore, patients with homozygous deletion or mutations of the IGF-1gene demonstrate prenatal and postnatal growth failure (31–34). Experimental evidence suggests that IGF-1 modulates bone growth primarily through its paracrine action in the growth plate, rather than by acting systemically. In mice with the concomitant disruption of the liver-specific Igf1 and of the acid labile subunit (Als) gene, circulating concentrations of IGF-1 are markedly reduced (85%–90% lower than the wild-type mice), and yet these transgenic animals’ femoral length is reduced only 20% compared to that of wild-type mice (35). Thus, these findings suggest that systemic IGF-1 has only a marginal role in inducing longitudinal bone growth. Of note, serum GH in these double knock-out mice is dramatically increased (⬃ 15 times), thus implicating a direct effect of GH on the long

Figure 6. Effects of systemic GH and IGF-1 on JAK2, STAT5B, and Akt in the tibial growth plate of KO and C mice. Threeweek old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of the treatment period, protein extracted from the tibial growth plates was electrophoresed and immunoblotted with antibodies directed to phosphorylated and total proteins. A representative blot from three independent experiments is presented for each protein.


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bone’s growth plate by increasing the expression and action of IGF-1 locally and/or by acting via IGF-1-independent mechanisms. Several lines of evidence suggest IGF-1-independent growth-promoting effects of GH. GH causes more robust longitudinal bone growth than IGF-1 in animals, and the effects of these factors may be additive (36 –38); in addition, administration of recombinant human (rh) GH has greater growth-promoting effects than those of recombinant IGF-1 in humans (39). The evidence supporting the fact that GH and IGF-1 promote longitudinal bone growth in part independently is consistent with the hypothesis that GH may have growth-promoting effects even in the absence of IGF-1 (21, 22). Another insulin-like growth factor, IGF2, has growthpromoting properties prenatally. Mice with deletion of Igf2 exhibit growth retardation at birth (their birth size is similar to that of Igf1 null mice); they also exhibit small placentas (40, 41). When both Igf1 and Igf2 genes are deleted in mice, their birth size is more significantly re-

Figure 7. Effects of systemic GH and IGF-1 on BMP-2 mRNA expression and on NF-␬B p65 mRNA expression and protein phosphorylation in the tibial growth plate of KO and C mice. Three-week old KO and C mice (n ⫽ 4 –7/group) were untreated or treated with daily injections of GH or IGF-1 for 4 weeks. At the end of the experimental period, total RNA and protein were extracted from the tibial growth plate of KO and C mice. mRNA expression was quantitated by real-time PCR. The relative expression levels of mRNA were normalized by ␤-actin in the same samples. Results were expressed as fold change compared to untreated control (mean ⫾S.E). Extracted proteins were electrophoresed and immunoblotted with antibodies directed to phosphorylated and total proteins. A representative blot from three independent experiments is presented for each protein.

Endocrinology

duced compared to that of mice with either Igf1 or Igf2 deletion, thus supporting a distinctive, rather than redundant, role for each of these two genes on fetal growth. Unlike Igf1 null mice, Igf2 null mice do not experience postnatal growth retardation. On the other hand, Igf2 is still abundantly expressed in the rodent growth plate in the first few weeks of postnatal life, to progressively diminish afterwards (42). Interestingly, Igf2 mRNA expression is increased in Igf1 null mice and reduced in Ghr null mice (21); thus, GH could, even in the absence of Igf1 expression in the growth plate, promote chondrogenesis by stimulating Igf2 expression. Consistent with these findings, in

Figure 8. In vitro effects of GH and IGF-1 on cell proliferation and collagen X mRNA expression of cultured chondrocytes transfected with Igf1r siRNA and/or Ghr siRNA. Chondrocytes were transfected with control siRNA, Igf1r siRNA, and/or Ghr siRNA. Cells were then cultured in the absence or presence of IGF-1 (100ng/ml) or GH (10ng/ml) for 24 hours. A: At the end of culture period, 2.5 ␮Ci/well of 3H-thymidine (Amersham) was added to the culture medium for an additional 3 hours. Chondrocytes were released by trypsin and collected onto glass fiber filters. Incorporation of 3H-thymidine was measured by liquid scintillation counting. Results are expressed as % of control and represent mean values obtained from three independent experiments. B: Total RNA was extracted from chondrocytes and then processed as described in Materials and Methods. Collagen X mRNA expression was determined by real time PCR. The relative expression levels of mRNA were normalized by ␤-actin in the same samples. Results were expressed as fold change compared to control chondrocytes (mean ⫾S.E).


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our study we have demonstrated an increased Igf2 mRNA expression in the growth plate of mice (both WT and KO) injected with GH, compared to untreated mice. The type 1 IGF receptor, more commonly known as IGF1 receptor, binds both IGF1 and IGF2 with high affinity (43). The mitogenic and metabolic actions of IGF1 and IGF2 are mediated through the IGF1 receptor; in fact, monoclonal antibodies blocking the binding of IGF1 or IGF2 to the IGF1 receptor prevent the ability of both IGFs to stimulate cell replication (44, 45). Deletion of Igf1r in mice leads to a 45% reduction of birth size, with 100% neonatal lethality (46). Concurrent deletion of Igf1 and Igf1r results in no further reduction of birth size compared to that following deletion of Igf1r alone, thus indicating that virtually all the effects of IGF1 on body growth are mediated by the IGF1 receptor (47). Unlike IGF1, part of the promoting effect of IGF2 on embryonic growth appears to be mediated by the insulin receptor (48). Several patients with IGF1R gene mutations/deletions have been reported (49). Although the phenotype may vary, many of these patients exhibit severe intrauterine growth retardation (IUGR) and postnatal growth failure. Thus far, the most severe postnatal growth retardation (height – 7.3 SDS) was reported in a subject with a compound heterozygous mutation of the IGF1R gene (50). In recent years, a number of children with IGF1R mutations have been treated with GH: in most of these children, GH induced a detectable, although modest, growth response (49). Consistent with our findings in the mouse growth plate, IGF-independent effects of GH have also been demonstrated in other tissues and organs. Treatment with GH, but not IGF-1, in humans significantly increased serum concentrations of mannan-binding lectin, a component of the innate immune system (51). Systemic infusion of GH in humans increased forearm blood flow without significantly increasing plasma IGF-1 levels, muscle IGF-1 expression, or Akt phosphorylation (52). Furthermore, GH facilitates muscle growth independent of IGF-1 effects (53). Lastly, IGF-1R-independent effects of GH have been also demonstrated in cultured osteoblasts (54). In our study, KO mice exhibited a reduced tibial growth plate height, which was due to reduced height of all three zones (epiphyseal, proliferative, and hypertrophic). In addition, the systemic injection of GH in KO mice for 4 weeks led to an increase of the growth plate height, with such effect resulting from the increased height of the epiphyseal, proliferative, and hypertrophic zones. In cultured chondrocytes transfected with IGF-1R siRNA, the addition of GH stimulated both thymidine incorporation and collagen X mRNA expression. Thus, our findings suggest that GH has IGF-dependent and IGF-independent effects


9

on both chondrocyte proliferation and differentiation. A tamoxifen-inducible, cartilage-specific Igf1r knockout mouse has previously been made and studied by Wang Y et al (55). These investigators examined the transgenic mice’ tibial growth plate 7– 8 days after induction of gene excision by tamoxifen; consistent with our findings, they observed decreased chondrocyte proliferation and reduced hypertrophic zone height. However, this study did not evaluate either the animals’ bone growth overtime or the effects of systemic GH on bone growth or growth plate chondrogenesis. In contrast to our findings, in another study (21) on the global deletion of Igf1 in mice the authors described an enlarged growth plate epiphyseal zone. Chondrocyte numbers and proliferation were not significantly different in the growth plate of the Igf1 null and littermate WT mice, while chondrocyte hypertrophy was significantly reduced in the Igf1 null mice. The authors hypothesized that the spared chondrocyte proliferation in Igf1 null mice may have resulted from their increased circulating GH levels, which would induce (directly or via increased expression of Igf2 in the growth plate) cell proliferation in the growth plate. In contrast, the normal serum GH levels of our KO mice with a targeted postnatal deletion of Igf1r in the growth plate would prevent any compensatory effects on the reduced chondrocyte proliferation resulting from the lack of IGF1 and IGF2 effects in the growth plate. In our study, the systemic injection of GH induced the phosphorylation of JAK2 in the KO mouse tibial growth plate, thus indicating that GH promoted tibial growth by specifically binding and activating GHR. With respect to the IGF-independent molecular mechanisms through which GH may affect growth plate chondrogenesis and bone growth, we speculated that NF-␬B p65 (an intracellular transcription factor) may be a mediator of the growth-promoting effects of GH. We have previously demonstrated that NF-␬B p65 promotes chondrocyte proliferation and differentiation (56). In a subsequent study, we have also shown that GH activates NF-␬B p65 in cultured chondrocytes, and its promoting effects on chondrocyte proliferation and differentiation are diminished after silencing NF-␬B p65 expression (57). Lastly, we have reported a child with a mutation of the I␬B␣ gene (and secondary NF-␬B p65 impaired action) and GH insensitivity (58). In the present study, the injection of GH in KO mice resulted in an increased phosphorylation (ie, activation) of NF-␬B p65 in the growth plate. In contrast, the injection of IGF-1 in KO mice did not modify NF-␬B p65 phosphorylation. Thus, this evidence suggests that NF-␬B p65 may be one of the intracellular transcription factors mediating the IGF-independent growth-promoting effects of GH in our tamoxifen-inducible, cartilage-specific Igf1r


10


knockout mice. Regarding additional factors modulating the IGF-independent effects of GH, published evidence indicates that Bone Morphogenetic Protein-2 (BMP-2) may also have such mechanistic role. We have previously shown that BMP-2, which is expressed in the growth plate, accelerates longitudinal bone growth by stimulating growth plate chondrocyte proliferation and chondrocyte hypertrophy (59). Another study has shown that GH increases the expression of BMP-2 in fibroblasts in vitro (60). In this study, our findings indicate that systemic GH induces BMP-2 mRNA expression in the growth plate of KO mice; in contrast, systemic IGF-1 does not modify BMP-2 mRNA expression. Thus, these data suggest that the GH-dependent increased expression of BMP-2 in the growth plate may be responsible, at least in part, of the IGF-independent, growth-promoting effects of GH. We are aware of two major limitations of our study. First, the systemic effects of GH and IGF-1 described by us may not necessarily mimic the physiological effects of these two hormones in mammalian bone growth. Second, although IGF-1 and IGF-2 exert most of their biological effects by binding and activating IGF-1R we cannot rule out that they may affect growth plate chondrogenesis and bone growth through other receptors (ie, insulin receptor). However, the lack of Akt phosphorylation in the growth plate of GH-treated or IGF-1-treated KO mice would argue against a significant activation of the insulin receptor by IGFs. In conclusion, our findings indicate that GH can modulate growth plate chondrogenesis and longitudinal bone growth through IGF-1R-independent mechanisms in the growth plate. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.

Acknowledgments Shufang Wu was supported in part by grants from the National Natural Science Foundation of China (No.81071440/H0601 and No. 81 472 038) and National Natural Science Foundation of China for Excellent Young Scientist (No. 81 222 026). This research was supported in part by Novo Nordisk. We thank Dr. Susan Mackem for providing a pair of mice expressing a CreERCart transgene. Address all correspondence and requests for reprints to: Corresponding author and person to whom reprint requests should be addressed: Francesco De Luca, M.D.,St. Christopher’s Hospital for Children, 3601 A Street, Philadelphia, PA 19 134.Phone: 215– 427-8101. Fax: 215– 427-8105. E-mail: [email protected]. Disclosure Summary: the authors have nothing to disclose

Endocrinology

This work was supported by.

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