Novel late response genes of PTHrP in chondrocytes.

To gain more insight into the downstream effectors of parathyroid hormone (PTH) related peptide (PTHrP) signaling in chondrocytes, we performed microarray analysis to identify late PTHrP response genes using the chondrogenic ATDC5 cell line and studied their response in the osteoblastic KS483 cell line and explanted metatarsals. At day 8 of micromass culture, ATDC5 cells have pre-hypertrophic-like characteristics and at this time point the cells were stimulated with PTHrP for 24 and 72 h and RNA was isolated. PTHrP treatment inhibited outgrowth of cartilage matrix and decreased the expression of Col10a1 mRNA, which is in line with the inhibitory effects of PTHrP on chondrocyte differentiation. Using cDNA microarray analysis, a list of 9 genes (p< 10(-3)) was generated, including 3 upregulated (IGFBP4, Csrp2, and Ecm1) and 6 downregulated (Col9a1, Col2a1, Agc, Hmgn2, Calm1, and Mxd4) response genes. Four out of 9 genes are novel PTHrP response genes and 2 out of 9 have not yet been identified in cartilage. Four out of 9 genes are components of the extra-cellular matrix and the remaining genes are involved in signal transduction and transcription regulation. The response to PTHrP was validated by quantitative PCR, using the same RNA samples as labeled in the microarray experiments and RNA samples isolated from a new experiment. In addition, we examined whether these genes also reacted to PTHrP in other PTHrP responsive models, like KS483 osteoblasts and explanted metatarsals. The expression of late PTHrP response genes varied between ATDC5 chondrocytes, KS483 osteoblasts and metatarsals, suggesting that the expression of late response genes is dependent on the cellular context of the PTHrP responsive cells.

Multiple mechanisms are involved in inhibition of osteoblast differentiation by PTHrP and PTH in KS483 Cells.

UNLABELLED: We examined the mechanism by which PTHrP and PTH inhibit KS483 osteoblastic differentiation. We show that PTHrP and PTH inhibit differentiation downstream of early BMP signaling and downregulated components of the hedgehog (Hh) signaling cascade. In addition, PTHrP and PTH repressed RunX2 and osx expression. Overexpression of either gene, however, could not relieve PTHrP and PTH's inhibitory actions. Our data suggest that multiple parallel mechanisms are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH. INTRODUCTION: PTH-related peptide (PTHrP) and PTH are potent inhibitors of osteoblast differentiation in vitro by as yet unexplained mechanisms. MATERIALS AND METHODS: We treated murine bone marrow stromal cells and the mesenchymal progenitor cell line KS483 with PTHrP and PTH in combination with either BMPs or hedgehog (Hh) and measured early and late markers of osteoblast differentiation and studied the expression of RunX2 and Osterix (osx). In addition, we examined the PTHrP and PTH response in stable KS483 cells overexpressing either RunX2 or osx. RESULTS: PTHrP and PTH inhibited BMP- and Hh-induced osteogenesis downstream of early BMP signaling and by downregulation of components of the Hh signaling cascade. PTHrP and PTH prevented the upregulation of RunX2 expression associated with osteoblast differentiation in an indirect response. However, PTHrP and PTH could still inhibit differentiation, and particularly matrix mineralization, of cells expressing RunX2. In addition, PTHrP and PTH potently downregulated osx expression only in mature osteoblasts in an intermediate early response, but osx overexpression could not relieve the inhibitory effects of PTHrP and PTH on matrix mineralization. CONCLUSIONS: Our data suggest that, besides transcriptional repression of RunX2 and osx, other mechanisms in parallel with or downstream of RunX2 and osx are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH in vitro.

Downregulation of Wnt signaling by increased expression of Dickkopf-1 and -2 is a prerequisite for late-stage osteoblast differentiation of KS483 cells.

UNLABELLED: We examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. It has been shown that Wnt signaling in mature osteoblasts needs to be downregulated to enable the formation of a mineralized matrix. Using RNA interference, we showed that this is, at least in part, accomplished by upregulation of the Wnt antagonists Dickkopf-1 and -2. INTRODUCTION: The role of Wnt signaling in the initiation of osteoblast differentiation has been well studied. However, the role during late-stage differentiation is less clear. We have examined the role of Wnt/beta-catenin signaling in successive stages of osteoblast differentiation. MATERIALS AND METHODS: We treated murine bone marrow and mesenchymal stem cell-like KS483 cells with either LiCl or Wnt3A during several stages of osteoblast differentiation. In addition, we generated stable KS483 cell lines silencing either the Wnt antagonist Dkk-1 or -2 RESULTS: Activation of Wnt signaling by LiCl inhibits the formation of a mineralized bone matrix in both cell types. Whereas undifferentiated KS483 cells respond to Wnt3A by inducing nuclear beta-catenin translocation, differentiated cells do not. This is at least in part accomplished by upregulated expression of Dkk-1 and -2 during osteoblast differentiation. Using RNA interference, we showed that Dkk-1 plays a crucial role in blunting the BMP-induced alkaline phosphatase (ALP) response and in the transition of an ALP+ osteoblast in a mineralizing cell. In contrast, Dkk-2 plays a role in osteoblast proliferation and the initiation of osteoblast differentiation. CONCLUSIONS: Our data suggest that Wnt signaling in maturing osteoblasts needs to be downregulated to enable the formation of a mineralized bone matrix. Furthermore, they suggest that Dkk-1 and Dkk-2 may have distinct functions in osteoblast differentiation.

Hedgehog stimulates only osteoblastic differentiation of undifferentiated KS483 cells.

The involvement of hedgehog signaling in the initiation of osteoblastic differentiation in the bone collar during endochondral bone formation has been well established. The stages at which hedgehog acts during osteoblast differentiation as well as its molecular mechanism of action are less well understood. To address these questions, we have made use of the preosteoblastic cell line KS483. First, a systematic survey of mRNA expression of osteoblastic differentiation showed expression of Ihh and signaling intermediates at all stages. Interestingly, expression of Ihh, Gli1 and Ptc1 peaked during the maturation phase. Addition of recombinant human sonic hedgehog (rShh) potently increased osteoblastic differentiation of KS483 cells dose-dependently as assayed by a modest increase in alkaline phosphatase (ALP) activity, a strong increase in matrix mineralization, and increased mRNA expression of established osteoblast marker genes. These effects were blocked by the hedgehog antagonist cyclopamine, which by itself was ineffective. Addition of rShh during early stages was sufficient, while addition to mature osteoblasts had no effect. Furthermore, hedgehog signaling could be completely blocked by the BMP antagonists, soluble truncated BMPR-IA and noggin. In contrast, the BMP-induced differentiation of KS483 cells could only be partly inhibited by high doses of cyclopamine. These data demonstrate that Hh-induced osteoblastic differentiation requires functional BMP signaling. In KS483 cells, Hh and BMP synergistically induced alkaline phosphatase activity only when suboptimal concentrations of BMP were used. This synergy did not occur at the level of immediate early BMP response, but at the level of Hh response as determined by transient transfection studies using either a BMP reporter or a Gli reporter construct. In addition, rShh inhibited adipogenesis of KS483 cells cultured under adipogenic culture conditions, suggesting that Hh is involved in directing differentiation of KS483 cells toward osteoblasts at the expense of adipogenesis. Using in situ hybridization, we demonstrated, for the first time, Ihh mRNA expression in vivo in osteoblasts and lining cells in the humerus of developing human skeleton. Our in vitro and in vivo data indicate a stimulatory role for osteoblast-expressed Ihh in bone formation in a positive feedback loop. It may recruit progenitor cells in the osteoblastic lineage at the expense of adipocytes and it may stimulate maturation of early osteoblasts.