XBP1S Associates with RUNX2 and Regulates Chondrocyte Hypertrophy

BMP2 (bone morphogenetic protein 2) is known to activate unfolded protein response signaling molecules, including XBP1S and ATF6. However, the influence on XBP1S and ATF6 in BMP2-induced chondrocyte differentiation has not yet been elucidated. In this study, we demonstrate that BMP2 mediates mild endoplasmic reticulum stress-activated ATF6 and directly regulates XBP1S splicing in the course of chondrogenesis. XBP1S is differentially expressed during BMP2-stimulated chondrocyte differentiation and exhibits prominent expression in growth plate chondrocytes. This expression is probably due to the activation of the XBP1 gene by ATF6 and splicing by IRE1a. ATF6 directly binds to the 5′-flanking regulatory region of the XBP1 gene at its consensus binding elements. Overexpression of XBP1S accelerates chondrocyte hypertrophy, as revealed by enhanced expression of type II collagen, type X collagen, and RUNX2; however, knockdown of XBP1S via the RNAi approach abolishes hypertrophic chondrocyte differentiation. In addition, XBP1S associates with RUNX2 and enhances RUNX2-induced chondrocyte hypertrophy. Altered expression of XBP1S in chondrocyte hypertrophy was accompanied by altered levels of IHH (Indian hedgehog) and PTHrP (parathyroid hormone-related peptide). Collectively, XBP1S may be a novel regulator of hypertrophic chondrocyte differentiation by 1) acting as a cofactor of RUNX2 and 2) affecting IHH/PTHrP signaling.     

Regulation of chondrocyte differentiation by IRE1α depends on its enzymatic activity

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1α (IRE1α),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1α in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1α inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1α-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1α C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin–epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1α expression in chondrogenesis. The expression of IRE1α is depended on GEP signaling, and IRE1α expression is hardly detectable in GEP^−/− embryos. In addition, IRE1α inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1α in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1α may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.     

Aggrecan modulation of growth plate morphogenesis

Chick and mouse embryos with heritable deficiencies of aggrecan exhibit severe dwarfism and premature death, demonstrating the essential involvement of aggrecan in development. The aggrecan-deficient nanomelic (nm) chick mutant E12 fully formed growth plate (GP) is devoid of matrix and exhibits markedly altered cytoarchitecture, proliferative capacity, and degree of cell death. While differentiation of chondroblasts to pre-hypertrophic chondrocytes (IHH expression) is normal up to E6, the extended periosteum expression pattern of PTCH (a downstream effector of IHH) indicates altered propagation of IHH signaling, as well as accelerated down-regulation of FGFR3 expression, decreased BrdU incorporation and higher levels of ERK phosphorylation, all indicating early effects on FGF signaling. By E7 reduced IHH expression and premature expression of COL10A1 foreshadow the acceleration of hypertrophy observed at E12. By E8, exacerbated co-expression of IHH and COL10A1 lead to delayed separation and establishment of the two GPs in each element. By E9, increased numbers of cells express P-SMAD1/5/8, indicating altered BMP signaling. These results indicate that the IHH, FGF and BMP signaling pathways are altered from the very beginning of GP formation in the absence of aggrecan, thereby inducing premature hypertrophic chondrocyte maturation, leading to the nanomelic long bone growth disorder.     

The canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner

To better understand the role of the canonical Wnt signaling pathway in cartilage development, we adenovirally expressed a constitutively active (ca) or a dominant negative (dn) form of lymphoid enhancer factor-1 (LEF-1), the main nuclear effector of the pathway, in undifferentiated mesenchymal cells, chondrogenic cells, and primary chondrocytes, and examined the expression of markers for chondrogenic differentiation and hypertrophy. caLEF-1 and LiCl, an activator of the canonical pathway, promoted both chondrogenic differentiation and hypertrophy, whereas dnLEF-1 and the gene silencing of beta-catenin suppressed LiCl-promoted effects. To investigate whether these effects were dependent on Sox9, a master regulator of cartilage development, we stimulated Sox9-deficient ES cells with the pathway. caLEF-1 and LiCl promoted both chondrogenic differentiation and hypertrophy in wild-type, but not in Sox9-deficient, cells. The response of Sox9-deficient cells was restored by the adenoviral expression of Sox9. Thus, the canonical Wnt signaling pathway promotes chondrocyte differentiation in a Sox9-dependent manner.     

Wnt5a and Wnt5b exhibit distinct activities in coordinating chondrocyte proliferation and differentiation

Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. We have studied the roles of two members of the Wnt family, Wnt5a and Wnt5b in long bone development. We show that Wnt5a is required for longitudinal skeletal outgrowth and that both Wnt5a and Wnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) negative feedback loop. We find that important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, we show that Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. Our data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones.     

p107 and p130 Coordinately regulate proliferation,Cbfa1 expression,and hypertrophic differentiation during endochondral bone development

During endochondral bone development, both the chondrogenic differentiation of mesenchyme and the hypertrophic differentiation of chondrocytes coincide with the proliferative arrest of the differentiating cells. However, the mechanisms by which differentiation is coordinated with cell cycle withdrawal, and the importance of this coordination for skeletal development, have not been defined. Through analysis of mice lacking the pRB-related p107 and p130 proteins, we found that p107 was required in prechondrogenic condensations for cell cycle withdrawal and for quantitatively normal alpha1(II) collagen expression. Remarkably, the p107-dependent proliferative arrest of mesenchymal cells was not needed for qualitative changes that are associated with chondrogenic differentiation, including production of Alcian blue-staining matrix and expression of the collagen IIB isoform. In chondrocytes, both p107 and p130 contributed to cell cycle exit, and p107 and p130 loss was accompanied by deregulated proliferation, reduced expression of Cbfa1, and reduced expression of Cbfa1-dependent genes that are associated with hypertrophic differentiation. Moreover, Cbfa1 was detected, and hypertrophic differentiation occurred, only in chondrocytes that had undergone or were undergoing a proliferative arrest. The results suggest that Cbfa1 links a p107- and p130-mediated cell cycle arrest to chondrocyte terminal differentiation.