CDC5L promotes early chondrocyte differentiation and proliferation by modulating pre-mRNA splicing of SOX9, COL2A1, and WEE1

Abstract

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process. Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process. Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition characterized by ectopic bone formation within the spinal posterior longitudinal ligament (PLL) in the spinal canal; this compresses the spinal cord and nerve root to cause severe myelopathy and root dysfunction (1Tsuyama N. Ossification of the posterior longitudinal ligament of the spine.Clin. Orthop. Relat. Res. 1984; : 71-84PubMed Google Scholar). OPLL affects 1.9–4.3% of the Japanese population, 0.4–3.0% of other Asian populations, and 0.1–1.7% of the Caucasian population (2Matsunaga S. Sakou T. Ossification of the posterior longitudinal ligament of the cervical spine: Etiology and natural history.Spine. 2012; 37: E309-E314Crossref PubMed Scopus (178) Google Scholar). To date, surgical treatment that decompresses the spinal cord is the only option to relieve severe neuropathy; however, surgical outcomes are often unsatisfactory because of irreversible spinal nerve damage resulting from long-term compression. Therefore, there is an urgent need to develop an alternative nonsurgical approach to prevent or control OPLL growth. Histological studies have indicated that OPLL is formed mainly through a process similar to endochondral ossification (3Sato R. Uchida K. Kobayashi S. Yayama T. Kokubo Y. Nakajima H. Takamura T. Bangirana A. Itoh H. Baba H. Ossification of the posterior longitudinal ligament of the cervical spine: Histopathological findings around the calcification and ossification front.J. Neurosurg. Spine. 2007; 7: 174-183Crossref PubMed Scopus (40) Google Scholar, 4Hashizume Y. Pathological studies on the ossification of the posterior longitudinal ligament (OPLL).Acta Pathol. Jpn. 1980; 30: 255-273PubMed Google Scholar, 5Liao C.C. Lee S.T. Symptomatic ossification of the posterior longitudinal ligament of the lumbar spine. Case report.J. Neurosurg. 1999; 91: 230-232PubMed Google Scholar, 6Sugita D. Yayama T. Uchida K. Kokubo Y. Nakajima H. Yamagishi A. Takeura N. Baba H. Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.Spine. 2013; 38: E1388-E1396Crossref PubMed Scopus (23) Google Scholar); we had observed the cartilage-specific type II and XI collagen expression in the cartilage-like area adjacent to the ossification front (7Maeda S. Ishidou Y. Koga H. Taketomi E. Ikari K. Komiya S. Takeda J. Sakou T. Inoue I. Functional impact of human collagen α2(XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine.J. Bone Miner Res. 2001; 16: 948-957Crossref PubMed Scopus (62) Google Scholar). In addition, we recently confirmed this concept through immunohistochemically evaluating the expression of a series of proteins specific to endochondral ossification around the ossification area of OPLL (8Maeda S. Kawamura I. Tominaga H. Taniguchi N. Histopathological features of ossification of the posterior longitudinal ligament.in: OPLL: Ossification of the Posterior Longitudinal Ligament. Springer, Singapore2020: 39-46Crossref Google Scholar). Endochondral bone formation is initiated by condensation of mesenchymal progenitor cells expressing the chondrogenic master transcription factor Sox9, which drives the expression of cartilage-specific matrix proteins such as type II collagen (COL II, Col2a1) and aggrecan (Agc1) in chondrocytes (9Akiyama H. Chaboissier M.C. Martin J.F. Schedl A. de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6.Genes Dev. 2002; 16: 2813-2828Crossref PubMed Scopus (1286) Google Scholar). Resting (nonproliferating) chondrocytes, specifically expressing parathyroid-hormone-related protein (PTHrP), are skeletal stem cells that undergo chondrocyte differentiation and proliferation (10Mizuhashi K. Ono W. Matsushita Y. Sakagami N. Takahashi A. Saunders T.L. Nagasawa T. Kronenberg H.M. Ono N. Resting zone of the growth plate houses a unique class of skeletal stem cells.Nature. 2018; 563: 254-258Crossref PubMed Scopus (128) Google Scholar). PTHrP maintains chondrocytes in a proliferative state, which is indispensable for endochondral bone growth (11Amizuka N. Warshawsky H. Henderson J.E. Goltzman D. Karaplis A.C. Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation.J. Cell Biol. 1994; 126: 1611-1623Crossref PubMed Scopus (413) Google Scholar, 12Karaplis A.C. Luz A. Glowacki J. Bronson R.T. Tybulewicz V.L. Kronenberg H.M. Mulligan R.C. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.Genes Dev. 1994; 8: 277-289Crossref PubMed Scopus (971) Google Scholar). As the distance from PTHrP-expressing resting chondrocytes increases, the columnar chondrocytes stop proliferating and differentiate into type X collagen (COL X, Col10a1)-expressing chondrocytes, chondrocytes and to The cartilage matrix to for bone formation H.M. of the growth PubMed Scopus Google Scholar). to date, the that chondrogenesis in spinal and cause OPLL is to be to OPLL or K. K. S. T. Ikari K. Koga H. Komiya S. Nakajima T. S. Inoue I. for genes of ossification of the posterior longitudinal ligament of the spine.J. Bone Miner Res. 2002; PubMed Scopus Google Scholar, A. I. S. H. Y. S. in in a of ossification of the posterior longitudinal ligament of the PubMed Scopus Google Scholar, Takahashi A. T. T. Baba H. Uchida K. S. A. S. K. Taniguchi Y. S. A. Nakajima H. genome-wide association study susceptibility loci for ossification of the posterior longitudinal ligament of the PubMed Scopus Google Scholar, T. Nakajima Ikari K. T. K. Uchida K. Y. H. N. S. T. K. of on Ossification of the genome-wide analysis of ossification of the posterior longitudinal ligament of the spine.J. Bone Miner 2013; PubMed Scopus Google Scholar). of the on the ossification of spinal including our have a genome-wide association study in the Japanese population to six susceptibility loci for OPLL: and Takahashi A. T. T. Baba H. Uchida K. S. A. S. K. Taniguchi Y. S. A. Nakajima H. genome-wide association study susceptibility loci for ossification of the posterior longitudinal ligament of the PubMed Scopus Google Scholar). is which of the genes within the loci result in protein that to the pathogenesis of OPLL, endochondral In this we on of the CDC5L division cycle because our analysis has to be expressed human and fibroblasts Takahashi A. T. T. Baba H. Uchida K. S. A. S. K. Taniguchi Y. S. A. Nakajima H. genome-wide association study susceptibility loci for ossification of the posterior longitudinal ligament of the PubMed Scopus Google Scholar). CDC5L is a pre-mRNA splicing factor and a of the human that an essential role in and into in the cell cycle of and Biol. PubMed Scopus Google Scholar, R. that proteins are required for pre-mRNA S. A. 1999; PubMed Scopus Google Scholar, B. K. Functional analysis of the human CDC5L and of by J. PubMed Scopus Google Scholar, E. I. A. W. H. R. of the human Cell Biol. 30: PubMed Scopus Google Scholar, A. A. I. W. of protein a of pre-mRNA splicing and Biol. PubMed Scopus Google Scholar). CDC5L promotes the expression of a of genes in and and the damage and as the pre-mRNA splicing efficiency of these genes was by CDC5L knockdown R. Y. W. T. B. N. T. N. of pre-mRNA splicing factor and PubMed Scopus Google Scholar). As a cell cycle CDC5L is in including Y. K. J. R. J. R. C.C. cycle gene a target for in Res. PubMed Scopus Google Scholar, S. expression and as for in PubMed Scopus Google Scholar, H. W. W. H. J. Y. R. T. and role of as a cell cycle protein in PubMed Scopus Google and roles W. W. Y. J. S. J. of CDC5L PubMed Scopus Google Scholar, Y. W. promotes of through of the PubMed Scopus Google Scholar, W. H. R. Y. W. of in cells on the Res. 2018; PubMed Scopus Google Scholar). the expression of CDC5L in OPLL or cartilage has there are the impact of CDC5L on cell Here, we CDC5L expression in OPLL and in bone cartilage We the roles of CDC5L in early chondrocyte differentiation and proliferation by pre-mRNA splicing of Sox9, Col2a1, and to the polymorphism adjacent to the CDC5L gene in Takahashi A. T. T. Baba H. Uchida K. S. A. S. K. Taniguchi Y. S. A. Nakajima H. genome-wide association study susceptibility loci for ossification of the posterior longitudinal ligament of the PubMed Scopus Google affects CDC5L expression in human we an expression loci analysis human the for the OPLL was in our specimens, cells CDC5L expression of an We CDC5L is expressed at the protein in human OPLL CDC5L protein was in cells We have characterized the area adjacent to the ossification front of OPLL in which the cells and II X, that these cells are in the early chondrocyte differentiation (8Maeda S. Kawamura I. Tominaga H. Taniguchi N. Histopathological features of ossification of the posterior longitudinal ligament.in: OPLL: Ossification of the Posterior Longitudinal Ligament. Springer, Singapore2020: 39-46Crossref Google Scholar). In this we found CDC5L expression and II and As CDC5L is a cell cycle we cells proliferative of the proliferating cell We found that these area cells We for CDC5L and and found a of in the area cells The cartilage area is characterized by the of chondrocytes, which are for and II which is the matrix calcification that the cells are in the endochondral ossification (8Maeda S. Kawamura I. Tominaga H. Taniguchi N. Histopathological features of ossification of the posterior longitudinal ligament.in: OPLL: Ossification of the Posterior Longitudinal Ligament. Springer, Singapore2020: 39-46Crossref Google As because chondrocytes CDC5L was in the cartilage area We Cdc5l protein expression endochondral ossification from Cdc5l was only in the columnar proliferating chondrocytes, as also in the chondrocytes in the cells in the proliferating state, as by and of a in OPLL and bone the roles of CDC5L in the early of endochondral To the role of Cdc5l in chondrocyte growth as a G2/M transition we cell proliferation assay Cdc5l siRNA transfection in the chondrogenic ATDC5 cell primary chondrocytes, and primary mesenchymal stem cells Cdc5l knockdown suppressed the growth of the cells To this was to G2/M we ATDC5 cells at the cells into the cell cycle by and The control cells to the cell in the G2/M and into cells the of the G2/M and in the G2/M that cell growth was at least in G2/M transition. We the expression of Wee1, the G2/M transition inhibitor A. S. K. H. gene in human cells.Nature. 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Nakajima H. genome-wide association study susceptibility loci for ossification of the posterior longitudinal ligament of the PubMed Scopus Google Scholar). as an for CDC5L gene we observed a role of Cdc5l in chondrocyte growth in CDC5L is in pre-mRNA splicing to the expression of genes R. Y. W. T. B. N. T. N. of pre-mRNA splicing factor and PubMed Scopus Google the target genes essential for the G2/M transition process are Cell cycle is by the of and cell cycle proteins essential for transition to the cell cycle formation S. and cell cycle 2013; PubMed Scopus Google Scholar). The to on the of the G2/M and and a to part of the factor J. J. factor the of a of the cell cycle control gene PubMed Scopus Google Scholar, J. J. T. is a of factor from PubMed Scopus Google Scholar). is by which of on H. of the by the human PubMed Scopus Google Scholar). is indispensable for the of chondrocyte proliferation in bone growth plate Takeda S. A. H. The indispensable role of in skeletal PubMed Scopus Google Scholar). findings are in our which showed that expression of Wee1, was in proliferating chondrocytes expression in the Cdc5l and Wee1 showed As Cdc5l cell growth was by Wee1 knockdown Wee1 is to be a target of Cdc5l in the G2/M transition. Cdc5l knockdown suppressed the cell CDC5L in ATDC5 chondrocytes also cell growth by CDC5L expression Wee1 expression In to cell into of the G2/M through which cells a that to the A. S. K. H. gene in human cells.Nature. PubMed Scopus Google Scholar, H. of the by the human PubMed Scopus Google Scholar, Wee1 cell division by on J. PubMed Scopus Google Scholar). the G2/M cells damage to to undergo to as D. T. Res. PubMed Scopus Google Scholar). a inhibitor of and in cells D. D. S. a Wee1 2012; PubMed Scopus Google Scholar). Therefore, is to be a at least in of the expression of the cell the condition in which the cells Wee1 expression and the expression of CDC5L and be to early chondrocyte OPLL susceptibility gene identified by was to chondrocyte differentiation of ATDC5 cells by the of the was in human fibroblasts I. H. S. of the on the Ossification of and of as a susceptibility gene for ossification of the posterior longitudinal ligament of the J. PubMed Scopus Google Scholar). Therefore, the common from and CDC5L is the of early which the that OPLL is formed through endochondral the that Cdc5l enhanced both proliferation and differentiation of chondrocytes is because cells the cell cycle undergo loss of the proliferation and the differentiation of chondrocytes Takeda S. A. H. The indispensable role of in skeletal PubMed Scopus Google and of Sox9 and Col2a1 expression in ATDC5 cells J. J. J. V.L. chondrocyte proliferation and differentiation through PubMed Scopus Google Scholar). the of CDC5L as a Cdc5l was indispensable for the pre-mRNA splicing of early chondrogenic genes in chondrocytes. Cdc5l is the unique protein that drives both early chondrocyte differentiation and As roles in the endochondral ossification process in to be cartilage-specific Cdc5l mice be in the In to chondrogenic loss of Cdc5l in enhanced expression of the ligament cell and and splicing and Wee1 splicing and expression also by Cdc5l knockdown and that CDC5L only promotes pre-mRNA splicing also in a was that transfection of CDC5L siRNA in genes decreased and genes R. Y. W. T. B. N. T. N. of pre-mRNA splicing factor and PubMed Scopus Google that CDC5L both and roles in pre-mRNA Therefore, specific of CDC5L in the that the splicing of or in to an splicing CDC5L as a transcription factor to target gene expression Y. W. promotes of through of the PubMed Scopus Google Scholar, W. H. R. Y. W. of in cells on the Res. 2018; PubMed Scopus Google Scholar, J. N. R. W. W. Y. W. W. CDC5L promotes expression and PubMed Scopus Google Scholar). Therefore, be that we to the of CDC5L as a transcription factor in The expression of CDC5L in cells in OPLL is to the ligament and the chondrogenic for the of endochondral The that be in cells in OPLL be in the OPLL or CDC5L may be in the chondrogenesis of because CDC5L pre-mRNA splicing of a of is an the and CDC5L in the be identified as In we expression in proliferating chondrocytes in OPLL or bone growth and or of the pre-mRNA splicing of early chondrogenic genes and Col2a1) or Wee1, roles of CDC5L in cell proliferation and differentiation the of endochondral the to in the expression loci analysis analysis was by the at the We human OPLL from was from the study was by the of studies by the of by the and of cells cultured in was to the was from the was a The of CDC5L expression was to in the was from fibroblasts the was by a assay Y. T. K. R. H. Y. for genome-wide association 2001; PubMed Scopus Google Scholar). The gene expression to the and the of an of was and a from at in in to be in The of human OPLL or and to by was by in The cell was in on the by for The primary II and X or as for primary a of and was to the was the of human OPLL or and for and for CDC5L and or by for or for by for of CDC5L and Cell and Cell of the The chondrogenic cell ATDC5 was from The cells in and The cell was from the which in and chondrocytes from cartilage of the and was and by for by The chondrocytes through a cell and cultured in and from and cultured in growth expression analysis of differentiation was as I. S. K. T. Ishidou Y. Komiya S. of chondrocytes by growth and bone protein Biol. 2012; PubMed Scopus Google Scholar). chondrogenic was at a of for or primary chondrocytes, growth factor was at and for siRNA a of of Cdc5l Wee1 and the control siRNA from The into cells The cell was the human CDC5L and cells by the of and by The cell was and and was to the The was the and the was the ATDC5 cells or at a of of cells the to and of was into the the was The of gene by analysis II and the was in and the expression of gene was to that of was on the and the by for analysis by the The in are in in and the and to the the primary Cell or the was or Cell the was and the are the indicated the cells to cell proliferation assay to the The was at on a cells and in for at in for cells in cells at in and at for in for cells for The cells at to and The cells at for at least and by and cell cycle was a bone rudiments from at or and cultured in and of the cultured to the the The control and Cdc5l shRNA from ATDC5 cells the cells cultured in in the was and of at a of cells by the or to the by and The the was and cells cultured for for In bone by of or in and The the and cultured in or was by the assay the in of ATDC5 cell or and for and to immunoprecipitation a or control to the and to and the was as ATDC5 cells or and for by to the and to The pre-mRNA splicing inhibitor was in and at and The are expressed as the of at least the was data required for the are within the The that have of the of this and by the of the on the Ossification of We for We from for a of this study was by from the for and and for the of for and S. and S. 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