1α,25-Dihydroxyvitamin D3 Inhibits GH-induced Expression of SOCS-3 and CIS and Prolongs Growth Hormone Signaling via the Janus Kinase (JAK2)/Signal Transducers and Activators of Transcription (STAT5) System in Osteoblast-like Cells
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m204819200
ISSN1083-351X
AutoresOrlando Morales, Malin Hedengran Faulds, Urban Lindgren, Lars-Arne Haldosén,
Tópico(s)Thyroid Disorders and Treatments
ResumoGrowth hormone (GH) and 1α,25-dihydroxyvitamin D3(1,25-(OH)2D3) are regulators of bone growth and bone metabolism. In target cells, GH activates several signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and STAT5a and b. The effects of 1,25-(OH)2D3are mediated via a nuclear receptor, the vitamin D receptor, which, when bound by 1,25-(OH)2D3, activates the transcription of target genes. In earlier studies (Morel, G., Chavassieux, P., Barenton, B., Dubois, P. M., Meunier, P. J., and Boivin, G. (1993) Cell Tissue Res.273, 279–286) synergistic interaction between 1,25-(OH)2D3and GH regarding expression of osteoblastic markers has been described. The UMR 106 cell line is a rat osteosarcoma cell line with osteoblast-like properties. We have recently shown (Morales, O., Lindgren, U., and Haldosen, L. A. (2000) J. Bone Miner. Res.15, 2284–2290) that UMR 106 cells express a GH-responsive JAK2/STAT5 signaling system. These cells also express the vitamin D receptor and respond to 1,25-(OH)2D3. In the present study we have investigated whether 1,25-(OH)2D3 influences GH signaling via the JAK2/STAT5 pathway in UMR 106 cells. We found that 1,25-(OH)2D3 prolonged GH signaling via the JAK2/STAT5 pathway. Pretreatment of cells with 1,25-(OH)2D3 was also necessary in order to detect GH-induced STAT5 transcriptional response. Furthermore, the pretreatment of cells with 1,25-(OH)2D3rendered to the cells the capacity to respond to repetitive GH-stimulation. In UMR 106 cells, GH induced the expression of the JAK/STAT negative regulatory proteins SOCS-3 and CIS. Interestingly, pretreatment with 1,25-(OH)2D3inhibited GH-induced expression of these proteins. From these results we propose that 1,25-(OH)2D3 has an inhibitory effect on negative regulatory pathways acting on JAK2 and/or STAT5 in UMR 106 cells and that this, in all or partly, explains the effects of 1,25-(OH)2D3 on GH-signaling via the JAK/STAT pathway. Growth hormone (GH) and 1α,25-dihydroxyvitamin D3(1,25-(OH)2D3) are regulators of bone growth and bone metabolism. In target cells, GH activates several signaling pathways, among them the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. GH mainly activates JAK2 and STAT5a and b. The effects of 1,25-(OH)2D3are mediated via a nuclear receptor, the vitamin D receptor, which, when bound by 1,25-(OH)2D3, activates the transcription of target genes. In earlier studies (Morel, G., Chavassieux, P., Barenton, B., Dubois, P. M., Meunier, P. J., and Boivin, G. (1993) Cell Tissue Res.273, 279–286) synergistic interaction between 1,25-(OH)2D3and GH regarding expression of osteoblastic markers has been described. The UMR 106 cell line is a rat osteosarcoma cell line with osteoblast-like properties. We have recently shown (Morales, O., Lindgren, U., and Haldosen, L. A. (2000) J. Bone Miner. Res.15, 2284–2290) that UMR 106 cells express a GH-responsive JAK2/STAT5 signaling system. These cells also express the vitamin D receptor and respond to 1,25-(OH)2D3. In the present study we have investigated whether 1,25-(OH)2D3 influences GH signaling via the JAK2/STAT5 pathway in UMR 106 cells. We found that 1,25-(OH)2D3 prolonged GH signaling via the JAK2/STAT5 pathway. Pretreatment of cells with 1,25-(OH)2D3 was also necessary in order to detect GH-induced STAT5 transcriptional response. Furthermore, the pretreatment of cells with 1,25-(OH)2D3rendered to the cells the capacity to respond to repetitive GH-stimulation. In UMR 106 cells, GH induced the expression of the JAK/STAT negative regulatory proteins SOCS-3 and CIS. Interestingly, pretreatment with 1,25-(OH)2D3inhibited GH-induced expression of these proteins. From these results we propose that 1,25-(OH)2D3 has an inhibitory effect on negative regulatory pathways acting on JAK2 and/or STAT5 in UMR 106 cells and that this, in all or partly, explains the effects of 1,25-(OH)2D3 on GH-signaling via the JAK/STAT pathway. 1α,25-dihydroxyvitamin D3(1,25-(OH)2D3) 1The abbreviations used are: 1, 25-(OH)2D3, 1α,25-dihydroxyvitamin D3; GH, growth hormone; GHR, GH receptor; bGH, bovine GH; IGF-1, insulin-like growth factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology 2; SOCS, suppressors of cytokine signaling; CIS, cytokine-inducible SH2-containing protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TTBS, TBS plus Tween 20; GEMSA, gel electrophoresis mobility shift assay. 1The abbreviations used are: 1, 25-(OH)2D3, 1α,25-dihydroxyvitamin D3; GH, growth hormone; GHR, GH receptor; bGH, bovine GH; IGF-1, insulin-like growth factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology 2; SOCS, suppressors of cytokine signaling; CIS, cytokine-inducible SH2-containing protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TTBS, TBS plus Tween 20; GEMSA, gel electrophoresis mobility shift assay. is the most active metabolite of vitamin D3 and is involved in several processes including Ca2+ transport, cell differentiation, immunological responses, and the regulation of gene expression (1Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). The effects of 1,25-(OH)2D3 are dependent upon the interaction of 1,25-(OH)2D3with a cytosolic/nuclear receptor, the vitamin D receptor (VDR), followed by the interaction of the steroid receptor complex with selective regions of the promoter of genes, which are either activated or repressed (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Vitamin D deficiency is associated with rickets in children and osteomalacia in adults. Earlier studies have shown that the effects of 1,25-(OH)2D3 on bone are mediated via the osteoblast (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 1,25-(OH)2D3is involved in differentiation of bone marrow stem cells into osteoclasts and osteoblast-like cells into osteoblasts. Differentiation into osteoblasts is accompanied by changes in biochemical properties of the cells such as, for example increased alkaline phosphatase activity and the increased synthesis of osteocalcin and type 1 collagen. VDR is present in normal osteoblast-like cells, osteosarcoma cells with osteoblast characteristics (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 3Langub M.C. Reinhardt T.A. Horst R.L. Malluche H.H. Koszewski N.J. Bone (NY). 2000; 27: 383-387Crossref PubMed Scopus (46) Google Scholar), and osteoclasts (3Langub M.C. Reinhardt T.A. Horst R.L. Malluche H.H. Koszewski N.J. Bone (NY). 2000; 27: 383-387Crossref PubMed Scopus (46) Google Scholar, 4Mee A.P. Hoyland J.A. Braidman I.P. Freemont A.J. Davies M. Mawer E.B. Bone (NY). 1996; 18: 295-299Crossref PubMed Scopus (65) Google Scholar).Growth hormone (GH) and insulin-like growth factor (IGF-1) are important regulators of longitudinal bone growth. GH regulates production of IGF-1 both in liver and bone cells. Recent data (5Le Roith D. Bondy C. Yakar S. Liu J.L. Butler A. Endocr. Rev. 2001; 22: 53-74Crossref PubMed Scopus (859) Google Scholar) indicate that the GH-regulated local production of IGF-1 in bone cells determines bone growth. Although many effects of GH on bone growth are mediated by IGF-1, experimental data (6Ohlsson C. Bengtsson B.A. Isaksson O.G. Andreassen T.T. Slootweg M.C. Endocr. Rev. 1998; 19: 55-79Crossref PubMed Scopus (712) Google Scholar) indicate that GH can directly influence bone cell function.GH activates several signaling pathways, among them the Janus kinase (JAK)/the signal transducers and activators of transcription (STAT) pathway (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). When GH binds to its receptor (GHR), it induces receptor homodimerization and activation of the GHR-associated tyrosine kinase Janus kinase 2 (JAK2) (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). This leads to the phosphorylation of JAK2 and intracellular proteins, including the GH receptor and the STATs. Upon phosphorylation, the STAT proteins either homodimerize or heterodimerize, translocate to the nucleus, bind to their appropriate DNA response element, and stimulate transcription of GH-regulated genes (8Herrington J. Smit L.S. Schwartz J. Carter-Su C. Oncogene. 2000; 19: 2585-2597Crossref PubMed Scopus (225) Google Scholar). At present, there are seven known mammalian members of the STAT gene family. GH has been demonstrated to activate STATs 1, 3, 5a, and 5b (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). Signaling pathways involved in the GH and IGF-1 effects on bone are, at present, not well understood, although it has been suggested that STAT5 mediates GH effects on bone growth (9Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (821) Google Scholar, 10Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). Activation of the GHR by GH initiates also negative regulatory pathways important for termination of GH signaling (11Ram P.A. Waxman D.J. J. Biol. Chem. 1999; 274: 35553-35561Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). One negative regulatory pathway may involve tyrosine phosphatases such as SHP-1 and SHP-2, which bind to phosphorylated residues on GHR, JAK2, and STAT5 via their SH2 domains and dephosphorylate and inactivate these molecules. A second negative regulatory pathway requires protein synthesis. GH induces the expression of the suppressors of cytokine signaling (SOCS) and the cytokine-inducible SH2-containing protein (CIS). These proteins act as a negative feedback loop by binding via their SH2 domains to phosphorylated JAK2 or GHR and inhibiting signaling (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar).We and others (12Morales O. Lindgren U. Haldosen L.A. J. Bone Miner. Res. 2000; 15: 2284-2290Crossref PubMed Scopus (9) Google Scholar, 13Gerland K. Bataille-Simoneau N. Basle M. Fourcin M. Gascan H. Mercier L. Mol. Cell. Endocrinol. 2000; 168: 1-9Crossref PubMed Scopus (21) Google Scholar) have recently shown that the osteoblast-like osteosarcoma cell line UMR 106 expresses a GH-responsive JAK2/STAT5 signaling system. In UMR 106 cells, GH activates JAK2 and STAT5a and b. These cells also have been shown to express VDR and to respond to 1,25-(OH)2D3 (14Forrest S.M., Ng, K.W. Findlay D.M. Michelangeli V.P. Livesey S.A. Partridge N.C. Zajac J.D. Martin T.J. Calcif. Tissue Int. 1985; 37: 51-56Crossref PubMed Scopus (165) Google Scholar). In some earlier studies (15Chenu C. Valentin-Opran A. Chavassieux P. Saez S. Meunier P.J. Delmas P.D. Bone (NY). 1990; 11: 81-86Crossref PubMed Scopus (132) Google Scholar,16Morel G. Chavassieux P. Barenton B. Dubois P.M. Meunier P.J. Boivin G. Cell Tissue Res. 1993; 273: 279-286Crossref PubMed Scopus (54) Google Scholar) synergistic interaction between 1,25-(OH)2D3 and GH regarding the expression of alkaline phosphatase and osteocalcin has been described. The molecular mechanism(s) for these effects is not understood. In the present study we have investigated whether 1,25-(OH)2D3influences GH signaling via the JAK/STAT pathway in UMR 106 cells. We found that 1,25-(OH)2D3 prolonged GH signaling via the JAK2/STAT5 pathway. Pretreatment of cells with 1,25-(OH)2D3 was also necessary in order to detect the GH-induced transcriptional response as measured by the activation of the STAT5-responsive reporter gene. Furthermore, the pretreatment of cells with 1,25-(OH)2D3rendered to the cells the capacity to respond to repetitive GH-stimulation. In UMR 106 cells, GH induced the expression of SOCS-3 and CIS. Interestingly, the pretreatment of cells with 1,25-(OH)2D3 inhibited GH-induced expression of these proteins. It is possible that this, in all or partly, explains the effects of 1,25-(OH)2D3 on GH-signaling via the JAK/STAT pathway in UMR 106 cells.DISCUSSIONWe have investigated the influence of 1,25-(OH)2D3 on GH signaling via the JAK/STAT pathway in UMR 106 osteoblast-like osteosarcoma cells. We found that the pretreatment of cells with 1,25-(OH)2D3slightly enhanced and significantly prolonged GH-induced activation of both JAK2 and STAT5. 1,25-(OH)2D3 pretreatment was also necessary in order to detect GH-induced STAT5 transcriptional activity. We have also shown that pretreatment with 1,25-(OH)2D3 rendered to UMR 106 cells the capacity to respond to repetitive stimulation with GH. Furthermore, pretreatment with 1,25-(OH)2D3 delayed and reduced GH-induced expression of SOCS-3 and CIS.In a recent study by Gerland et al. (13Gerland K. Bataille-Simoneau N. Basle M. Fourcin M. Gascan H. Mercier L. Mol. Cell. Endocrinol. 2000; 168: 1-9Crossref PubMed Scopus (21) Google Scholar) it was shown that GH could activate JAK2 and STAT5, but not STAT5 transcriptional activity in UMR 106 cells. They speculated that the expression levels of signaling components probably were too low for GH-induced activation of gene expression. They could show that it was only in STAT5 or GH receptor-transfected UMR 106 cells that GH could induce a transcriptional response. In our study we could detect GH-induced STAT5 DNA binding activity in both untreated and 1,25-(OH)2D3-pretreated UMR 106 cells (Figs. 4and 5), but it was only in 1,25-(OH)2D3-pretreated cells that GH could induce a functional STAT5 response (Fig. 6). 1,25-(OH)2D3 pretreatment did not increase the expression of JAK2 or STAT5 (Figs. 1 B and 2 B). Thus, the expression levels of these two proteins cannot explain the effects of 1,25-(OH)2D3 on STAT5 functional capacity. The only differences from 1,25-(OH)2D3-untreated cells that we could detect in 1,25-(OH)2D3-pretreated cells were a slightly increased and significantly prolonged GH-induced phosphorylation of JAK2 and a slightly increased and significantly prolonged presence of GH-activated STAT5 with DNA binding capacity in the nuclear compartment. The latter could be due to the prolonged influx of activated STAT5 into the nuclear compartment because of extended JAK2 activation. Another explanation could be the decreased nuclear deactivation of STAT5 (i.e. dephosphorylation) either in combination with extended JAK2 activation or as a sole cause. From these results we propose that 1,25-(OH)2D3has an inhibitory action on negative regulatory pathways acting on JAK2 and STAT5 in UMR 106 cells.1,25-(OH)2D3 influence on negative regulatory pathways can also be proposed from the second part of our study in which we analyzed the effect of 1,25-(OH)2D3 on the JAK/STAT signaling in UMR 106 cells repetitively stimulated with GH. In rats, mice, and certain other species the growth hormone secretory pattern is sexually dimorphic (20Davey H.W. Wilkins R.J. Waxman D.J. Am. J. Hum. Genet. 1999; 65: 959-965Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In adult male rats, plasma GH profiles are characterized by plasma GH pulses of 1-h duration every 3.5–4 h, with a typical peak plasma concentration of 200 ng/ml and very low GH levels in between peaks (21Jaffe C.A. Ocampo-Lim B. Guo W. Krueger K. Sugahara I. DeMott-Friberg R. Bermann M. Barkan A.L. J. Clin. Invest. 1998; 102: 153-164Crossref PubMed Scopus (193) Google Scholar). Females exhibit more continuous plasma GH levels of about 20–40 ng/ml. Pulsatile GH secretion, which begins at puberty, is more effective than continuous GH in promoting weight gain and longitudinal bone growth (22Isaksson O.G. Eden S. Jansson J.O. Annu. Rev. Physiol. 1985; 47: 483-499Crossref PubMed Google Scholar). The mechanism by which a temporal plasma profile of GH regulates body growth is not well understood. The GH secretory pattern also regulates the sexually dimorphic expression of certain genes in liver (23Mode A. J. Reprod. Fertil. Suppl. 1993; 46: 77-86PubMed Google Scholar). Waxman et al. (24Waxman D.J. Ram P.A. Park S.H. Choi H.K. J. Biol. Chem. 1995; 270: 13262-13270Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) studied the intracellular mediator of the effects of plasma GH pulses on sexually dimorphic liver-expressed genes. Pulsatile but not continuous GH exposure activates liver STAT5b. Disruption of the STAT5b gene leads to an apparent GH pulse insensitivity of liver tissue associated with a loss of male-specific liver gene expression (9Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (821) Google Scholar). Furthermore, these mice exhibit a decrease in male characteristic pubertal body growth rate. Using a rat hepatocyte-derived cell line, Waxman and co-workers (25Gebert C.A. Park S.H. Waxman D.J. Mol. Endocrinol. 1999; 13: 38-56Crossref PubMed Scopus (73) Google Scholar) have studied in detail the molecular mechanisms regulating activation/deactivation and repetitive stimulation of JAK2/STAT5 pathway in liver cells. It was shown that a minimum 2.5-h GH-free period was required in between repetitive GH-pulses for STAT5 activation to occur. They could also show that a tyrosine phosphatase and a serine/threonine kinase, both of unknown identity, took part in resetting JAK/STAT signaling for subsequent rounds of GH activation. As described under "Results," UMR 106 cells, not pretreated with 1,25-(OH)2D3, responded to the first GH pulse but only weakly to the second and not at all to the third GH pulse, despite 2.5-h GH-free period in between pulses (Fig. 7 A). Thus, the first pulse induced a GH refractory state. This is in contrast to the study of GH-pulsed liver cells described above, wherein cells responded to GH pulses with 2.5-h GH-free periods between pulses. Interestingly, in 1,25-(OH)2D3-pretreated UMR 106 cells the second and third GH pulse induced STAT5 DNA binding activity that was lower than after the first pulse but clearly detectable (Fig. 7 B).The GHR/JAK2/STAT5-pathway is negatively regulated by deactivation mechanisms. These systems are activated as GH activates GHR and are important for the termination of GH signaling (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). One negative regulatory pathway involved in GH signaling includes certain members of a family of eight cytokine-inducible proteins named suppressors of cytokine signaling or cytokine-inducible SH2 containing protein (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). GH has, in different experimental systems, been shown to induce the expression of different combinations of SOCS 1–3 and CIS genes, whose products act as a negative feedback loop by binding via their SH2 domains to tyrosine-phosphorylated JAK2 or GHR and thus inhibiting signaling (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). Interestingly, transgenic mice constitutively expressing CIS have reduced body weight (26Matsumoto A. Seki Y. Kubo M. Ohtsuka S. Suzuki A. Hayashi I. Tsuji K. Nakahata T. Okabe M. Yamada S. Yoshimura A. Mol. Cell. Biol. 1999; 19: 6396-6407Crossref PubMed Scopus (217) Google Scholar) and, conversely, mice deficient in SOCS-2 exhibit a giant or high growth phenotype (27Horvat S. Medrano J.F. Genomics. 2001; 72: 209-212Crossref PubMed Scopus (96) Google Scholar, 28Metcalf D. Greenhalgh C.J. Viney E. Willson T.A. Starr R. Nicola N.A. Hilton D.J. Alexander W.S. Nature. 2000; 405: 1069-1073Crossref PubMed Scopus (400) Google Scholar). In different studies it has been shown that glucocorticoids strongly inhibit both basal and hormone-induced expression of SOCS-3 (29Paul C. Seiliez I. Thissen J.P. Le Cam A. Eur. J. Biochem. 2000; 267: 5849-5857Crossref PubMed Scopus (61) Google Scholar, 30Tollet-Egnell P. Flores-Morales A. Stavreus-Evers A. Sahlin L. Norstedt G. Endocrinology. 1999; 140: 3693-3704Crossref PubMed Google Scholar). We show here that pretreatment with 1,25-(OH)2D3, in UMR 106 cells delayed and decreased GH-induced expression of SOCS-3 and CIS proteins (Fig. 8,A and B). These data correlate to the prolonged nuclear presence of STAT5 (Fig. 3) and extended STAT5 DNA binding activity (Fig. 4 B). Both SOCS-3 and CIS have been shown to inhibit or decrease GH activation of STAT5 and STAT5-dependent transcriptional activity (11Ram P.A. Waxman D.J. J. Biol. Chem. 1999; 274: 35553-35561Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 31Hansen J.A. Lindberg K. Hilton D.J. Nielsen J.H. Billestrup N. Mol. Endocrinol. 1999; 13: 1832-1843Crossref PubMed Google Scholar, 32Karlsson H. Gustafsson J.A. Mode A. Mol. Cell. Endocrinol. 1999; 154: 37-43Crossref PubMed Scopus (30) Google Scholar, 33Ram P.A. Waxman D.J. J. Biol. Chem. 2000; 275: 39487-39496Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Thus, it is possible that the down-regulation of SOCS-3 and CIS by 1,25-(OH)2D3 renders to UMR 106 cells the capacity to transcriptionally respond to GH and may also explain the prolonged DNA binding activity of STAT5 and the increased responsiveness to GH when given intermittently.A second negative regulatory pathway is thought to involve tyrosine phosphatases such as SHP-1 and SHP-2, which bind to and dephosphorylate GHR, JAK2, and STAT5. In the case of STAT5, this dephosphorylation has been reported to occur both in the cytosol and in the nucleus (34Ram P.A. Waxman D.J. J. Biol. Chem. 1997; 272: 17694-17702Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 35Yu C.L. Jin Y.J. Burakoff S.J. J. Biol. Chem. 2000; 275: 599-604Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Reduced expression or activity of SHP-1 and/or SHP-2 by 1,25-(OH)2D3 could be one explanation for its effect on GH-induced JAK2/STAT5 tyrosine phosphorylation. To our knowledge, no report has demonstrated regulated expression of SHP-1 or SHP-2 genes and, thus, these phosphatases seem to be constitutively expressed. Still, it remains to be studied if 1,25-(OH)2D3 can inhibit or reduce the expression and/or activity of these two tyrosine phosphatases.The physiological connection to our findings is not known at present. In some earlier in vitro cell studies it has been shown that co-administration as compared with separate administration of 1,25-(OH)2D3 and GH was more effective in increasing the expression of osteoblastic marker genes (15Chenu C. Valentin-Opran A. Chavassieux P. Saez S. Meunier P.J. Delmas P.D. Bone (NY). 1990; 11: 81-86Crossref PubMed Scopus (132) Google Scholar, 16Morel G. Chavassieux P. Barenton B. Dubois P.M. Meunier P.J. Boivin G. Cell Tissue Res. 1993; 273: 279-286Crossref PubMed Scopus (54) Google Scholar). If this increased expression of marker genes also was due to 1,25-(OH)2D3-enhanced GH signaling via the JAK2/STAT5 pathway remains to be studied. Furthermore, it can also only be speculated whether the growth retardation seen in children with vitamin D3 deficiency is partly due to relative GH refractoriness of bone cells. 1α,25-dihydroxyvitamin D3(1,25-(OH)2D3) 1The abbreviations used are: 1, 25-(OH)2D3, 1α,25-dihydroxyvitamin D3; GH, growth hormone; GHR, GH receptor; bGH, bovine GH; IGF-1, insulin-like growth factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology 2; SOCS, suppressors of cytokine signaling; CIS, cytokine-inducible SH2-containing protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TTBS, TBS plus Tween 20; GEMSA, gel electrophoresis mobility shift assay. 1The abbreviations used are: 1, 25-(OH)2D3, 1α,25-dihydroxyvitamin D3; GH, growth hormone; GHR, GH receptor; bGH, bovine GH; IGF-1, insulin-like growth factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology 2; SOCS, suppressors of cytokine signaling; CIS, cytokine-inducible SH2-containing protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TTBS, TBS plus Tween 20; GEMSA, gel electrophoresis mobility shift assay. is the most active metabolite of vitamin D3 and is involved in several processes including Ca2+ transport, cell differentiation, immunological responses, and the regulation of gene expression (1Bouillon R. Okamura W.H. Norman A.W. Endocr. Rev. 1995; 16: 200-257Crossref PubMed Google Scholar). The effects of 1,25-(OH)2D3 are dependent upon the interaction of 1,25-(OH)2D3with a cytosolic/nuclear receptor, the vitamin D receptor (VDR), followed by the interaction of the steroid receptor complex with selective regions of the promoter of genes, which are either activated or repressed (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Vitamin D deficiency is associated with rickets in children and osteomalacia in adults. Earlier studies have shown that the effects of 1,25-(OH)2D3 on bone are mediated via the osteoblast (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). 1,25-(OH)2D3is involved in differentiation of bone marrow stem cells into osteoclasts and osteoblast-like cells into osteoblasts. Differentiation into osteoblasts is accompanied by changes in biochemical properties of the cells such as, for example increased alkaline phosphatase activity and the increased synthesis of osteocalcin and type 1 collagen. VDR is present in normal osteoblast-like cells, osteosarcoma cells with osteoblast characteristics (2Zhang R. Ducy P. Karsenty G. J. Biol. Chem. 1997; 272: 110-116Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 3Langub M.C. Reinhardt T.A. Horst R.L. Malluche H.H. Koszewski N.J. Bone (NY). 2000; 27: 383-387Crossref PubMed Scopus (46) Google Scholar), and osteoclasts (3Langub M.C. Reinhardt T.A. Horst R.L. Malluche H.H. Koszewski N.J. Bone (NY). 2000; 27: 383-387Crossref PubMed Scopus (46) Google Scholar, 4Mee A.P. Hoyland J.A. Braidman I.P. Freemont A.J. Davies M. Mawer E.B. Bone (NY). 1996; 18: 295-299Crossref PubMed Scopus (65) Google Scholar). Growth hormone (GH) and insulin-like growth factor (IGF-1) are important regulators of longitudinal bone growth. GH regulates production of IGF-1 both in liver and bone cells. Recent data (5Le Roith D. Bondy C. Yakar S. Liu J.L. Butler A. Endocr. Rev. 2001; 22: 53-74Crossref PubMed Scopus (859) Google Scholar) indicate that the GH-regulated local production of IGF-1 in bone cells determines bone growth. Although many effects of GH on bone growth are mediated by IGF-1, experimental data (6Ohlsson C. Bengtsson B.A. Isaksson O.G. Andreassen T.T. Slootweg M.C. Endocr. Rev. 1998; 19: 55-79Crossref PubMed Scopus (712) Google Scholar) indicate that GH can directly influence bone cell function. GH activates several signaling pathways, among them the Janus kinase (JAK)/the signal transducers and activators of transcription (STAT) pathway (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). When GH binds to its receptor (GHR), it induces receptor homodimerization and activation of the GHR-associated tyrosine kinase Janus kinase 2 (JAK2) (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). This leads to the phosphorylation of JAK2 and intracellular proteins, including the GH receptor and the STATs. Upon phosphorylation, the STAT proteins either homodimerize or heterodimerize, translocate to the nucleus, bind to their appropriate DNA response element, and stimulate transcription of GH-regulated genes (8Herrington J. Smit L.S. Schwartz J. Carter-Su C. Oncogene. 2000; 19: 2585-2597Crossref PubMed Scopus (225) Google Scholar). At present, there are seven known mammalian members of the STAT gene family. GH has been demonstrated to activate STATs 1, 3, 5a, and 5b (7Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell. Signal. 2001; 13: 599-616Crossref PubMed Scopus (220) Google Scholar). Signaling pathways involved in the GH and IGF-1 effects on bone are, at present, not well understood, although it has been suggested that STAT5 mediates GH effects on bone growth (9Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (821) Google Scholar, 10Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). Activation of the GHR by GH initiates also negative regulatory pathways important for termination of GH signaling (11Ram P.A. Waxman D.J. J. Biol. Chem. 1999; 274: 35553-35561Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). One negative regulatory pathway may involve tyrosine phosphatases such as SHP-1 and SHP-2, which bind to phosphorylated residues on GHR, JAK2, and STAT5 via their SH2 domains and dephosphorylate and inactivate th
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