Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1625-9

INTEGRATIVE PHYSIOLOGY

Checkpoint kinase Chk2 controls renal Cyp27b1 expression, calcitriol formation, and calcium-phosphate metabolism Hajar Fahkri & Bingbing Zhang & Abul Fajol & Nati Hernando & Bernat Elvira & Julia G. Mannheim & Bernd J. Pichler & Christoph Daniel & Kerstin Amann & Atsushi Hirao & Jillian Haight & Tak W. Mak & Florian Lang & Michael Föller

Received: 6 June 2014 / Revised: 29 September 2014 / Accepted: 1 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)2D3), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1α-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2deficient mice (chk2−/−) were compared to wild-type mice (chk2+/+). Transcript levels of renal 25-hydroxyvitamin D 1α-hydroxylase, Chk2, and IRF-1 were determined by RTH. Fahkri : B. Zhang : A. Fajol : B. Elvira : F. Lang : M. Föller (*) Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tübingen, Germany e-mail: [email protected] N. Hernando Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zürich, Zürich, Switzerland J. G. Mannheim : B. J. Pichler Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, University of Tübingen, Röntgenweg 13, 72076 Tübingen, Germany C. Daniel : K. Amann Department of Nephropathology, University Hospital Erlangen, Erlangen, Germany A. Hirao Division of Molecular Genetics, Center for Cancer and Stem Cell Research, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa, Japan J. Haight : T. W. Mak : M. Föller The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada

PCR; Klotho expression by Western blotting; bone density by μCT analysis; serum or plasma 1,25 (OH)2D3, PTH, and Cterminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25hydroxyvitamin D 1α-hydroxylase as well as serum 1,25 (OH)2D3 and FGF23 levels were significantly lower in chk2−/− mice compared to chk2+/+ mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2−/− mice than in c hk 2 + / + m i c e d e s p i t e h y p o p h os p h a t e m i a a n d normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25hydroxyvitamin D 1α-hydroxylase expression thereby impacting on calcium and phosphate metabolism. Keywords PTH/vit D/FGF23 . Calcium/phosphate metabolism . Klotho . ATM

Introduction Checkpoint kinase 2 (Chk2) is a ubiquitously expressed serine/threonine kinase that is considered to be the main effector kinase of ataxia telangiectasia mutated (ATM) [6, 15, 37, 43]. ATM is a protein kinase which shares similarity with PI3 kinase and is also expressed in all tissues [14]. In response to genotoxicity, ATM activates Chk2 [6, 15, 37]. Chk2 then mediates G2 phase arrest by inducing further downstream effectors [16]. It is expressed throughout the cell cycle and even found in quiescent cells [28]. Chk2-mediated activation of downstream effectors such as p53, Mdm2, or Brca1 results in DNA repair, cell cycle arrest or delay, transcription of genes, and apoptosis [6]. Chk2-deficient mice (Chk2−/−) are fertile, seemingly normal, and do not develop spontaneous tumors [6, 21].

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Among the transcription factors stimulated by ATMdependent signaling is interferon-regulating factor 1 (IRF-1) [33]. IRF-1 has been shown to stimulate expression of Cyp27b1 or 25-hydroxyvitamin D 1α-hydroxylase, the key enzyme for the synthesis of calcitriol (1,25 (OH)2D3) [41]. In the human kidney, 25-hydroxyvitamin D 1α-hydroxylase converting 25 (OH)D3 to 1,25 (OH)2D3 is expressed in Bowman’s capsule, the proximal tubule, the thick ascending limb of Henle’s loop, the distal convoluted tubule, the cortical and medullary part of the collecting ducts, and the papillary epithelia [49]. The expression pattern is similar in mouse kidney; however, the proximal tubule is the main site of increased 25-hydroxyvitamin D 1α-hydroxylase expression during phosphorus restriction [50]. Importantly, IRF-1 is also expressed in the kidney [40] including the proximal tubule [2, 11, 45]. 1,25 (OH)2D3 is a powerful stimulator of intestinal and renal calcium and phosphate transport [5, 17]. 25hydroxyvitamin D 1α-hydroxylase and thus renal formation of 1,25 (OH)2D3 is stimulated by PTH [24] and inhibited by FGF23, a hormone synthesized in bone [25, 26, 30, 32, 34, 46]. Transmembrane Klotho protein serves as co-receptor for FGF23 [25, 30, 34]. 1,25 (OH)2D3 inhibits PTH secretion from the parathyroid glands [39], induces Klotho expression [20], and stimulates FGF23 release from bone [27]. Apart from its inhibitory effect on 1,25 (OH) 2D3 formation, FGF23 in concert with Klotho decreases the reabsorption of phosphate in the kidney directly. Although Chk2 is the main effector of ATM signaling and ATM has been shown to regulate expression of IRF-1, a transcription factor enhancing the expression of 25hydroxyvitamin D 1α-hydroxylase, nothing is known about a role of Chk2 for 1,25 (OH)2D3 formation and mineral metabolism. Here, we explored whether Chk2 deficiency influences 1,25 (OH)2D3 serum levels and calcium and phosphate metabolism. To this end, Chk2-deficient mice (chk2−/−) were compared to wild-type mice (chk2+/+).

Real-time (RT)-PCR Chk2+/+ and chk2−/− mice were sacrificed, and kidney samples snap-frozen. Next, total RNA was isolated using peqGOLD TriFast (peqLAB Biotechnologie GmbH, Erlangen, Germany) reagent, a method which is based on a chloroform extraction protocol. Messenger RNA (mRNA) was transcribed with SuperScript III Reverse Transcriptase (Invitrogen, Karlsruhe, Germany) using an oligo dT primer. Quantitative RT-PCR was performed on a BioRad iCycler iQ™ Real-Time PCR Detection System (Bio-Rad Laboratories, München, Germany) using the following primers: Chk2: Forward (5′-3′): TGGCTCCTGAGGTTCTTG Reverse (5′-3′): GGACACTTGGGTCTTATGCT Cyp27b1 (25-hydroxyvitamin D 1α-hydroxylase): Forward (5′-3′): CAGTTTACGTTGCCGACCCTA Reverse (5′-3′): GGACAGTGACTTTCTTGTCGC Irf-1: Forward (5′-3′): GCACGGCTGGGACATCAA Reverse (5′-3′): GCTGTGGTCATCAGGTAGGGTA Slc34a1 [9, 31]: Forward (5′-3′): TGATCACCAGCATTGCCG Reverse (5′-3′): GTGTTTGCAAGGCTGCCG Slc34a3 [9, 31]: Forward (5′-3′): TAATCTTCGCAGTTCAGGTTGCT Reverse (5′-3′): CAGTGGAATTGGCAGTCTCAAG

Methods

Gapdh:

Mice

Forward (5′-3′): GGTGAAGGTCGGTGTGAACG Reverse (5′-3′): CTCGCTCCTGGAAGATGGTG

All animal experiments were conducted according to the German law for the welfare of animals and were approved by the authorities of the state of Baden-Württemberg. Experiments were performed in male gene-targeted mice lacking functional Chk2 (chk2−/−) and in age-matched wildtype mice (chk2+/+) at the age of 8–15 weeks. The generation of the mice (background: C57BL/6) has been described elsewhere [21]. The mice were maintained on standard chow containing 1 % calcium, 0.7 % phosphorus, and 1000 IU vitamin D3/kg (Ssniff, Soest, Germany).

The final volume of the PCR reaction mixture was 20 μl and contained 2 μl cDNA, 1 μM of each primer, 10 μl GoTaq qPCR Master Mix (Promega, Mannheim, Germany), and sterile water up to 20 μl. qPCR conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 58 °C for 30 s. Calculated mRNA expression levels were normalized to the expression levels of Gapdh of the same complementary DNA (cDNA) sample. Relative quantification of gene expression was performed using the ΔΔCt

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method. For the mRNA expression analysis in different organs (Fig. 1c), the final volume of the RT-PCR reaction mixture was 15 μl and contained 1 μl cDNA, 1 μM of each primer, 7.5 μl GoTaq Master Mix Green (Promega), and sterile water up to 15 μl. PCR conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 45 s. The product size was analyzed on a 2 % agarose gel. Cell culture and transcript analysis Human embryonic kidney cells (HEK293) were cultured in DMEM high glucose medium supplemented with 10 % FCS and 1 % penicillin/streptomycin under standard conditions. Cells were treated without or with Chk2 Inhibitor II hydrate (Sigma; 5 μM) for 24 h. Total RNA was isolated using peqGOLD TriFast. mRNA was transcribed with GoScript Reverse Transcriptase random hexamers. Quantitative RTPCR was performed on a BioRad iCycler iQTM Real-Time PCR Detection System using the following primers as described above: hCYP27B1: Forward (5′-3′): CAGACAAAGACATTCATGTGGG Reverse (5′-3′): GTTGATGCTCCTTTCAGGTAC

Western blotting Klotho protein abundance was determined in snap-frozen kidneys from chk2+/+ and chk2−/− mice. After homogenization in lysis buffer (54.6 mM HEPES, 2.69 mM Na4P2O7, 360 mM NaCl, 10 % (v/v) glycerol, 1 % (v/v) NP40 or RIPA lysis buffer (Cell Signaling, Frankfurt, Germany)) containing phosphatase and protease inhibitor cocktail tablet (Complete Mini, Roche, Mannheim, Germany), the samples were incubated on ice for 30 min and then centrifuged at 14,000 rpm and 4 °C for 20 min. The supernatant was removed and used for Western blotting. Total protein (80 μg) was separated by SDS-PAGE, thereafter transferred to nitrocellulose membranes and blocked in 5 % non-fat milk/Tris-buffered saline/Tween-20 (TBST) at room temperature for 1 h. Membranes were probed overnight at 4 °C with polyclonal rat anti-Klotho antibody (1:1000 in 5 % fat-free milk in TBST; kindly provided by Akiko Saito, Kyowa Hakko Kirin Co., Ltd., Japan). After incubation with horseradish peroxidase-conjugated anti-rat or anti-rabbit secondary antibodies (Cell Signaling, Frankfurt, Germany; 1:2000) for 1 h at room temperature, the bands were visualized with enhanced chemiluminescence reagents (Amersham, Freiburg, Germany). Membranes were also probed with GAPDH antibody as a loading control. Densitometric analysis was performed using Quantity One software (Bio-Rad, München, Germany). Determination of bone density by μCT analysis

hIRF-1: Forward (5′-3′): CAAATCCCGGGGCTCATCTGG Reverse (5′-3′): CTGGCTCCTTTTCCCCTGCTTTGT hGAPDH: Forward (5′-3′): GAGTCAACGGATTTGGTCGT Reverse (5′-3′): GACAAGCTTCCCGTTCTCAG

Fig. 1 Lower 25-hydroxyvitamin D 1α-hydroxylase expression and 1,25 (OH)2D3 serum levels in chk2−/− mice (a). Arithmetic means± SEM (n=12–13) of relative Cyp27b1 (encoding 25-hydroxyvitamin D 1α-hydroxylase) transcript levels in kidneys from Chk2-deficient mice (chk2−/−, black bar) and wild-type mice (chk2+/+, white bar) (b).

Bone density was determined by μCT analysis [10]. Mice were sacrificed, and the hind legs were removed and fixed in formalin. The samples were scanned with a high-resolution multimodality single photon emission computed tomography (SPECT)/CT scanner (Siemens Preclinical Solutions, Knoxville, TN, USA) using a transaxial field of view of 5.4 cm and an axial field of view of 3.6 cm. The X-ray tube parameters were set at 80 kVp and 500 μA. The images were

Arithmetic means±SEM (n=9) of the serum 1,25 (OH)2D3 concentration in Chk2-deficient mice (chk2−/−, black bar) and wild-type mice (chk2+/+, white bar) (c). Chk2 mRNA expression pattern in different tissues from a chk2+/+ and a chk2−/− mouse. *p

Checkpoint kinase Chk2 controls renal Cyp27b1 expression, calcitriol formation, and calcium-phosphate metabolism.

Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling...
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