Up-regulation of per mRNA Expression by Parathyroid Hormone through a Protein Kinase A-CREB-dependent Mechanism in Chondrocytes

Abstract

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage. In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage. Recent studies have revealed that the endogenous circadian rhythmicity generated at the cellular level by circadian core oscillators resides not only in the hypothalamic suprachiasmatic nucleus of the anterior hypothalamus (1Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1891) Google Scholar, 2Storch K.F. Lipan O. Leykin I. Viswanathan N. Davis F.C. Wong W.H. Weitz C.J. Nature. 2002; 417: 78-83Crossref PubMed Scopus (1252) Google Scholar), which is recognized as the mammalian central clock, but also in various peripheral tissues including heart (3Young M.E. Razeghi P. Taegtmeyer H. Circ. Res. 2001; 88: 1142-1150Crossref PubMed Scopus (217) Google Scholar), adipose tissue (4Shimba S. Ishii N. Ohta Y. Ohno T. Watabe Y. Hayashi M. Wada T. Aoyagi T. Tezuka M. Proc. Natl. Acad. Sci. U. S. 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In mice, the rhythmic transcription of two orthologs of the Drosophila Period (per) gene appears to be essential for circadian rhythms. Expression of mouse per (mper) genes is known to be positively regulated by other clock proteins belonging to the basic helix-loop-helix period/aryl hydrocarbon receptor nuclear translocator/single minded class, which are Clock and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), 2The abbreviations used are: Bmal1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1; ChIP, chromatin immunoprecipitation; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; Cry, cryptochrome; DIG, digoxigenin; M-CSF, macrophage colony stimulating factor; Per, Period; PKA, protein kinase A; PKC, protein kinase C; PPR, parathyroid hormone/parathyroid hormone-related peptide receptor; PTH, parathyroid hormone; RANKL, receptor activator nuclear factor-κB ligand; Runx2, runt-related transcription factor 2; ZT, zeitgeber time; FBS, fetal bovine serum; HEK, human embryonic kidney; RT, reverse transcriptase. respectively. In addition, mPer proteins constitute multimeric complexes with products of the cryptochrome (cry) genes, mcry1 and mcry2, which in turn negatively regulate the gene transcription mediated by Clock/Bmal1 (8Gekakis N. Staknis D. Nguyen H.B. Davis F.C. Wilsbacher L.D. King D.P. Takahashi J.S. Weitz C.J. Science. 1998; 280: 1564-1569Crossref PubMed Scopus (1566) Google Scholar, 9Yamaguchi S. Mitsui S. Miyake S. Yan L. Onishi H. Yagita K. Suzuki M. Shibata S. Kobayashi M. Okamura H. Curr. Biol. 2000; 10: 873-876Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 10Griffin E.A. Staknis D. Weitz C.J. Science. 1999; 286: 768-771Crossref PubMed Scopus (520) Google Scholar, 11Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). Bone is formed through a well organized and highly regulated process (12Karsenty G. Nature. 2003; 423: 316-318Crossref PubMed Scopus (343) Google Scholar). During embryogenesis mesenchymal precursor cells differentiate into skeletal elements by forming a cartilaginous model framework, which then induces the bone formation process known as endochondral ossification in the vertebral column and long bone (13Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2177) Google Scholar). This endochondral ossification is responsible for the formation of the cartilaginous rudiment that is a tightly regulated region in both the differentiation and maturation of chondrocytes. Within this cartilaginous rudiment, for example, chondrocytes successively differentiate through a well organized mechanistic procedure with migration from resting cells at the rudiment edge to proliferating, hypertrophic and calcifying cells around the central region, which finally leads to mineralization of the cartilage matrix in the area of the hypertrophic chondrocytes. Shortly after the mineralization process takes place, however, most hypertrophic chondrocytes undergo sustained apoptosis. Upon death of chondrocytes after mineralization, circulating precursors of osteoblasts, osteoclasts, and capillaries begin to invade the cartilage matrix to produce new bones, leading to the growth of endochondral bones (12Karsenty G. Nature. 2003; 423: 316-318Crossref PubMed Scopus (343) Google Scholar, 13Kronenberg H.M. Nature. 2003; 423: 332-336Crossref PubMed Scopus (2177) Google Scholar). Previous reports have demonstrated that expression patterns of two major extracellular matrices in bone, type I collagen and osteocalcin, display circadian rhythms (14Simmons D.J. Nichols G. Am. J. Physiol. 1966; 210: 411-418Crossref PubMed Scopus (59) Google Scholar, 15Gundberg C.M. Markowitz M.E. Mizruchi M. Rpsen J.F. J. Clin. Endocrinol. Metab. 1985; 60: 736-739Crossref PubMed Scopus (211) Google Scholar). Molecular clock components have been shown to mediate leptin-regulated bone formation (16Fu L. Patel M.S. Bradley A. Wagner E.F. Karsenty G. Cell. 2005; 122: 803-815Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar), whereas to date no evidence is available for a of the expression of circadian clock genes in the differentiation of chondrocytes in the parathyroid hormone (PTH) is well known to regulate a of cellular through protein in both and is that circadian clock genes may be to a in the with of cellular in chondrocytes as well as In the study, has been to the expression and of different circadian clock genes in chondrocytes to circadian oscillation of cartilage during endochondral the of the for and was by the for of at were to to the of and to to in at of were for 2 under and with a with to and were then to from femurs at the zeitgeber 6, and is as the time of and respectively. were used for time different MC3T3-E1 and chondrogenic ATDC5 cells were from MC3T3-E1 cells were in essential of were with and ATDC5 cells were at a of 1 in a of and of were with and bovine was from and femurs of and for h with macrophage factor at in essential for cells in were by and subsequent at for The was and in essential FBS, at and receptor activator of nuclear factor-κB at were at a of 1 by at under for was as L. E. A. T. Y. J. 2005; PubMed Scopus Google Scholar). The central metatarsal were from mouse at of was in a well of a 1 of essential with 1 and These were at in a for In was as L. E. A. T. Y. J. 2005; PubMed Scopus Google Scholar). In were with by and were with at for The were with with with at for and with for different were with PTH, by of 1 h after and subsequent of mRNA expression a and to mper2 to and mper3 to promoters and the promoters in of the cAMP-responsive element (CRE) within the and mper2 promoters were from The region to and first intron to and to of the mouse type II collagen were from mouse by the and for the region, and and for the first intron The products were into the basic clock, and and expression were by Reppert of were with a into or HEK293 cells and after cells were and activity was in a to the was by the activity of was as E. S. Y. J. 2003; PubMed Scopus Google Scholar). In was with the and reverse from was and products were into a for of the results by are by not was at with for the gene for products were by a by of the of expression of mRNA for gene that of used for in a new was as E. S. Y. J. 2003; PubMed Scopus Google Scholar). In nuclear were by the of E. P. Res. PubMed Scopus Google with were in and the addition of to a of cells were at for were in and for and were in for by at for The were as nuclear were core sequence for the from the promoter type II collagen promoter and type II collagen first intron as a by for of protein of nuclear was at a of of protein and by a were and by exposure to for different to for subsequent by a was as E. S. Y. J. 2003; PubMed Scopus Google Scholar). In nuclear from were with and were to a by to a The were then to with by with the protein or the were with the with and with the of for subsequent exposure to was as E. S. Y. J. 2003; PubMed Scopus Google Scholar). In was and positively of to the by were at for 1 and with a of at for were then and with by with and subsequent exposure to for to were to the with the ATDC5 cells. ATDC5 cells were with for by in 1 and 1 was with the by of with was in the region to and first intron region to of the mouse type II collagen in addition to the mper promoter to as are all as the and was by and or of with Expression of Clock and in Bone and mRNA expression for bone and cartilage genes rhythms in addition to circadian clock genes, was from mouse femurs h a 24-h by of the mRNA levels by shown in all the clock genes and examined in this exhibited h rhythmicity in mouse femurs in vivo. expression was at for at for and and at for respectively. The expression of mRNA for type I type II and type X collagen exhibited a 24-h rhythm with a peak at and a to no rhythmic expression was in mRNA for the or genes in mouse These genes either to be or to with mRNA expression of runt-related transcription factor 2 a 24-h rhythm with a peak at but no in mRNA expression. Expression of mRNA for Clock in Bone circadian clock genes are in bone cells, including osteoclasts, osteoblasts, and was for clock the of of of osteoblasts, cells were for in the of and RANKL, by of expression of clock revealed that expression of mRNA was increased for all the genes examined in from in the of both and mRNA was at of the differentiation with expression of mRNA 1 after differentiation by and However, no expression was seen for the other clock genes, including clock, and in the of differentiation in osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells were for in differentiation by of expression of circadian clock Expression of mRNA was seen for all clock genes examined in both MC3T3-E1 and ATDC5 cells for in addition to mouse hypothalamus used as a control Expression of mRNA for Clock in ATDC5 examined the expression of circadian clock genes in ATDC5 cells. ATDC5 cells are chondrogenic cells and display a in the of in to the of the period to Expression of mRNA was increased for type X collagen during of from to with a to whereas a level of mRNA expression was seen for both type II collagen and for a period of to expression of mRNA was for and clock from to with a to whereas mRNA levels were significantly for per1 and at expression of mRNA was increased from to with a sustained to was seen in mRNA for or examined to the sequence in ATDC5 cells for different to with the mRNA expression for both clock and activity of the was in to the of from to the nuclear proteins involved in was in nuclear from ATDC5 cells for the The was in of the was by the whereas with of the core sequence not at a of of PTH mRNA Expression in MC3T3-E1 and ATDC5 PTH induces per1 in and osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells were for by the addition of PTH and subsequent of at different time after the PTH addition for of the mRNA levels by mRNA levels were increased 1 h after the addition of with a to the with in 2 h in both MC3T3-E1 and ATDC5 cells whereas no expression was seen at time after the addition of not The addition of of at in ATDC5 cells with at 1 h and a to the level within whereas no was seen for the expression of at time after PTH addition to h the of different known to per in addition to PTH. ATDC5 cells were with a of mper promoter to the reporter by with different for the the activator and PTH a of promoter activity, with no by other These growth growth growth growth and In addition, only significantly mper2 promoter activity in addition to PTH, whereas mper3 promoter activity was not by of the examined not of PTH mPer cells were with either of the mper promoter and with PTH at for and mper2 promoter were significantly increased after exposure to PTH, with no for the mper3 promoter the signaling pathway responsible for and mper2 by PTH, cells were with the mper by with the protein kinase A inhibitor and the protein kinase inhibitor 1 h PTH of by of both the and mper2 promoters by PTH, whereas the inhibitor but significantly the promoter activity of without significantly affecting that of PTH significantly increased the activity of of The inhibitor of PKA, but not of PKC, reporter was in promoter including and CRE, in the of either or the element to PTH within the mper promoter region and to the and elements responsible for and mper2 promoter deletion were generated from the promoter to with and PTH significantly the reporter activity of promoter CRE, without that of the other promoters not These a with a with two and a with the of in the of the promoter to PTH, were at the within in the of and mper2 This the of both the and mper2 promoters to PTH. In addition, MC3T3-E1 cells with either of mper promoter were with PTH at or at promoter were significantly increased by PTH and whereas was by only for the mper2 In no was seen for the mper3 as shown in ATDC5 cells. the of promoter activity by PTH and was of the promoter were used not of PTH mRNA Expression in and PTH induces mper as seen in ATDC5 cells in vitro, mouse were by organotypic for and subsequent with PTH for in cells were by a for type I collagen known to be by in the not In type II collagen mRNA was by to prehypertrophic whereas mRNA was by prehypertrophic chondrocytes with highly expression of type X collagen mRNA by hypertrophic in addition to mRNA expression by hypertrophic chondrocytes In for was by prehypertrophic chondrocytes. the used mRNA levels for both the per1 and per2 by prehypertrophic chondrocytes at h with a to levels within h after the addition of However, to mper expression in at time after not In with no expression was for of the genes examined not the of mper by PTH in as seen in ATDC5 cells in and were with PTH, by of and subsequent in In revealed that of mRNA expression was in prehypertrophic as seen in 1 h after PTH mRNA expression was also increased by the of PTH in to bone in the of in II the of mPer in the expression of genes, promoter were to a The mouse type II collagen promoter a recognized by the whereas the first intron region of mouse type II collagen also has a In HEK293 cells with a of different promoter promoter activity was significantly enhanced by whereas no was in cells with or Clock In and significantly the of promoter In the in promoter activity by the mouse type II collagen promoter was activated by by the clock genes However, mouse type II collagen first intron reporter activity was significantly enhanced by expression in a manner sensitive to by the introduction of or as was also seen with promoter In not significantly the mouse type II collagen first intron reporter activity the reporter with the core sequence was used in HEK293 cells the of clock genes in type II collagen transcription in cells, mouse type II collagen first intron reporter to both the elements and was used in ATDC5 cells. In ATDC5 cells, mouse type II collagen first intron reporter to activity was significantly enhanced by whereas no was in cells with or Clock the association of with the intron of type II collagen in we ATDC5 cells shown in the association of Clock was in ATDC5 cells with within the type II collagen first in addition to the but not the type II collagen In no association of Clock was in ATDC5 cells with within the type II collagen first intron in addition to the mper The of to the in the promoter and first intron region of mouse type II collagen was by in HEK293 cells with was in nuclear of HEK293 cells transiently in the in the first but not in the of mouse type II collagen The essential of the is that PTH stimulates expression of both and mper2 mRNA through mediated by a signaling pathway in both chondrocytes and with by mPer of mouse type II collagen first intron reporter activity but not type II collagen promoter reporter rhythm was for the expression of osteoblastic and genes in mouse and in addition, a of PTH mRNA expression in prehypertrophic chondrocytes in the of in vivo. this is the first direct of the expression of clock genes by chondrocytes in of direct of cellular studies have demonstrated the functional expression of clock genes in peripheral including bone, no direct evidence for a of clock genes in the cellular of chondrocytes has been The is that is a of the of with in association with at different of the These protein for the of protein for the of and protein for the of H. J. P. H.M. A.B. PubMed Google Scholar, S. A. Endocrinol. 10: PubMed Google Scholar). Previous studies have demonstrated that may regulate the expression of various genes the of signaling in cells. The signaling pathway appears to regulate the expression of type X and for example, whereas the signaling pathway a in the mineralization and of in addition to and in chondrocytes and chondrogenic cells as ATDC5 cells E. M. T. S. Y. Y. Res. 2001; PubMed Scopus Google Scholar, X. L. Y. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. H. H.M. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, is that the of signaling activated by protein to as cell and differentiation in of and differentiation of chondrocytes S. D. PubMed Scopus Google Scholar, T. M. A. K. Suzuki Y. J. Clin. PubMed Scopus Google Scholar). signaling is with the of cell and in addition to signaling D. D. D. J. 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A revealed a which be to a within of the region and the first intron of the type II collagen The from reporter and however, suggest that the to the in the first intron but not that in the of the In addition to the mouse type II collagen gene the element is within the first intron region but not in the region of the type X collagen the type I collagen osteoblastic element in the of evidence that be mediated by the element in the intron but not that in the with to elements in the first and of are for circadian by the Clock protein Shearman L.P. Reppert S.M. U. 2000; Google Scholar), in the the receptor gene through region in the intron K. H. N. J. 2005; PubMed Scopus Google Scholar). the protein be into the nucleus to the activity of the (8Gekakis N. Staknis D. Nguyen H.B. Davis F.C. Wilsbacher L.D. King D.P. Takahashi J.S. Weitz C.J. Science. 1998; 280: 1564-1569Crossref PubMed Scopus (1566) Google Scholar), may be that the mPer transiently by PTH negatively the of genes by through the in the intron region in chondrocytes. In this study, we reporter activity HEK293 cells, which are a human embryonic cell with The mouse type II collagen first intron reporter used in HEK293 cells not elements in the However, the type II collagen first intron the was by of in ATDC5 cells. is that the in the mouse type II collagen first intron for subsequent regulation of and mPer proteins in turn negatively regulate the transcription mediated by in chondrogenic cells, as (8Gekakis N. Staknis D. Nguyen H.B. Davis F.C. Wilsbacher L.D. King D.P. Takahashi J.S. Weitz C.J. Science. 1998; 280: 1564-1569Crossref PubMed Scopus (1566) Google Scholar, 9Yamaguchi S. Mitsui S. Miyake S. Yan L. Onishi H. Yagita K. Suzuki M. Shibata S. Kobayashi M. Okamura H. Curr. Biol. 2000; 10: 873-876Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 10Griffin E.A. Staknis D. Weitz C.J. Science. 1999; 286: 768-771Crossref PubMed Scopus (520) Google Scholar, 11Kume K. Zylka M.J. Sriram S. Shearman L.P. Weaver D.R. Jin X. Maywood E.S. Hastings M.H. Reppert S.M. Cell. 1999; 98: 193-205Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). The as well as the functional of expression of clock genes during in maturation of chondrogenic ATDC5 cells, however, to be in appears circadian clock genes are by chondrocytes to regulate extracellular matrix in cartilage. Molecular clock signaling is a for the of a for the and of a of bone and cartilage to and maturation of chondrocytes as well as in human