Indian hedgehog signaling regulates transcription and expression of collagen type X via Runx2/Smads interactions

Abstract

Indian hedgehog (Ihh) is essential for chondrocyte differentiation and endochondral ossification and acts with parathyroid hormone-related peptide in a negative feedback loop to regulate early chondrocyte differentiation and entry to hypertrophic differentiation. Independent of this function, we and others recently reported independent Ihh functions to promote chondrocyte hypertrophy and matrix mineralization in vivo and in vitro. However, the molecular mechanisms for these actions and their functional significance are still unknown. We recently discovered that Ihh overexpression in chondrocytes stimulated the expression of late chondrocyte differentiation markers and induced matrix mineralization. Focusing on collagen type X (Col10α1) expression and transcription, we observed that hedgehog downstream transcription factors GLI-Krüppel family members (Gli) 1/2 increased COL10A1 promoter activity and identified a novel Gli1/2 response element in the 250-bp basic promoter. In addition, we found that Ihh induced Runx2 expression in chondrocytes without up-regulating other modulators of chondrocyte maturation such as Mef2c, Foxa2, and Foxa3. Runx2 promoted Col10α1 expression in cooperation with Ihh. Further analyses using promoter assays, immunofluorescence, and binding assays showed the interaction of Gli1/2 in a complex with Runx2/Smads induces chondrocyte differentiation. Finally, we could demonstrate that Ihh promotes in vitro matrix mineralization using similar molecular mechanisms. Our data provide an in vitro mechanism for Ihh signaling to positively regulate Col10α1 transcription. Thus, Ihh signaling could be an important player for not only early chondrocyte differentiation but maturation and calcification of chondrocytes.

Keywords: Calcification; Chondrocyte; Col10a1; Differentiation; Gli1/Gli2-responsive Element; Hedgehog Signaling Pathway; Maturation; Runx2; Smads; Transcription.

FIGURE 1.

Gene expression profile induced by Ihh in chondrogenic cells. A and B, Ihh was overexpressed in ATDC5 cells with or without treatment of 5 μm cyclopamine. Isolated RNA was subjected to qPCR analysis using primers as shown in the figures (A). **, p < 0.01 (versus GFP-treated group). ##, p < 0.01 (versus Ihh-treated group) (n = 3 or 4). Cell proliferation was assessed using WST-1 reagent (B). **, p < 0.01 (versus GFP-treated group) (n = 4). C, changes in gene expression of Col10a1 is shown in A and were confirmed at the protein level using Western blotting. IB, immunoblot. D, changes in mRNA expression of hedgehog signal molecules and Col10a1 shown in A were also confirmed upon treatment of cells with Ihh protein instead of Ihh virus. (n = 3).

FIGURE 2.

Identification of the Gli-binding site in the 250-bp basic promoter of COL10A1. A, 250- or 2500-bp luciferase constructs of COL10A1 promoter were transfected into ATDC5 with either empty vectors, Gli1, or Gli2 expression vectors, and the luciferase/Renilla value was measured 48 h after transfection. **, p < 0.01; *, p < 0.05 (versus control vector) (n = 3). B, 250-bp luciferase constructs of COL10A1 promoter were transfected into ATDC5 with either empty vectors or different amounts of Gli2 expression vectors, and the luciferase/Renilla value was measured 48 h after transfection. **, p < 0.01; *, p < 0.05 (versus control vector). #, p < 0.05 (versus Gli2–0.25 ng group) (n = 3). C, 1800-bp luciferase constructs of COL10A1 promoter were used in a similar luciferase assay described in A to confirm reaction of Gli2 on COL10A1 promoter in ATDC5 cells. *, p < 0.05 (versus control vector) (n = 3). D, sequence analysis of the 250-bp basic promoter region of COL10A1. The suggested Gli-binding site in this study is highlighted in red. Previously reported Runx2-binding site is shown in blue. Conventional Smad-binding elements are indicated in green. Underlined areas show either the long probes (65 bp) or short probes (29 bp) used in the oligonucleotide pulldown assay. Arrows harbor the fragments amplified in CHIP assay. E, oligonucleotide pulldown assay using 29-bp probe of WT and mutants. F, sequence comparing the WT and mutants of suggested Gli-binding site in the COL10A1 promoter. The 29-bp probes containing either Mut1 or Mut2 were used in E, and 250-bp luciferase constructs having the insertion of point mutations as either Mut3 or Mut4 were used in H. G, oligonucleotide pulldown assay using a 10-bp probe of the suggested Gli-binding site showing interaction with Gli1. H, luciferase assay was performed in COS7 cells using either WT or mutant 250-bp COL10A1 promoter as mentioned under “Experimental Procedures.” The data were shown as fold change when compared with the increase using a WT promoter vector. **, p < 0.01 (versus WT vector) (n = 3). IB, immunoblot.

FIGURE 3.

Ihh induces Runx2 expression but does not up-regulate other hypertrophic modulators. A and B, Ihh was overexpressed in primary chondrocytes. RNA was collected on the 2nd or 4th day of cultures and subjected to qPCR analyses of chondrogenic markers as mentioned in the figure. **, p < 0.01 (versus GFP-treated group). n = 3. C, cell lysates of primary chondrocytes treated with GFP or Ihh viruses were collected in the 4th days of cultures and subjected to Western blotting to observe the changes at protein levels. IB, immunoblot. D, primary chondrocytes treated with GFP or Ihh viruses were fixed and immunostained with anti-Foxa2 antibody in the 4th days of cultures. IF, immunofluorescence.

FIGURE 4.

Ihh positively controls Col10α1 expression in cooperation with Runx2 in chondrocytes. A and C, Runx2 or DN-Runx2 was overexpressed in ATDC5 as confirmed by the protein level using Western blotting. IB, immunoblot. B, Ihh was overexpressed in ATDC5 cells with or without Runx2 co-infection, and Col10α1 expression was measured by qPCR analyses. **, p < 0.01 (versus GFP-treated group). ##, p < 0.01 (versus Runx2 or Ihh-treated group) (n = 3). D, Ihh was overexpressed in ATDC5 with or without DN-Runx2 co-infection, and Col10α1 expression was measured by qPCR analyses. *, p < 0.05 (versus GFP-treated group). ##, p < 0.01 (versus Ihh-treated group) (n = 3). E, Runx2 was overexpressed in ATDC5 with or without treatment of cyclopamine (Cyclo), and Col10α1 expression was measured by qPCR analyses. **, p < 0.01 (versus GFP-treated group). ##, p < 0.01 (versus Runx2-treated group) (n = 3).

FIGURE 5.

Interaction of Gli1 and Runx2 in the regulation of Col X. A, 250-bp luciferase construct of COL10A1 promoter was transfected into COS7 with expression vectors as indicated in the figure, and the luciferase/Renilla value was measured after 48 h. **, p < 0.01 (versus control). ##, p < 0.01 (versus Runx2 or Gli1) (n = 4). B, physical interaction between Gli1 and Runx2 was assessed by co-IP. Samples were immunoprecipitated by FLAG and immunoblotted by Myc. C, Myc-Gli1 and Dsred-Runx2 were co-transfected into C3H10T1/2 cells, and cells were immunostained by α-Myc antibody. Nucleus staining was performed with DAPI. D and E, oligonucleotide pulldown assay using a 65-bp COL10A1 promoter probe. The representative data are shown here. IB, immunoblot.

FIGURE 6.

Co-localization of Gli1/2 and pSmads in BMP2-treated C3H10T1/2 cells. A and B, Myc-Gli1 or Gli2 were transfected into C3H10T1/2 cells that are followed by 100 ng/ml BMP2 stimulation. The samples were immunostained by α-Myc and α-pSmad antibody, and the nucleus was stained with DAPI. The representative data are shown here.

FIGURE 7.

Involvement of Smads in Col X regulation conducted by hedgehog signal. A, 2.5-kb luciferase construct of the COL10A1 promoter was transfected into COS7 with expression vectors as indicated in the figure, and the luciferase/Renilla value was measured after 48 h. **, p < 0.01 (versus control). ##, p < 0.01 (versus Gli1 or Smad1/4) (n = 3). B, 2.5-kb luciferase construct of COL10A1 promoter was transfected into C3H10T1/2 cells with expression vectors as indicated followed by 100 ng/ml recombinant BMP2, and the luciferase/Renilla value was measured after 48 h. *, p < 0.05 (versus control). #, p < 0.05 (versus Gli2 or Smad1) (n = 3). C and D, physical interaction between Gli1/2 and Smad1 was assessed by co-IP. Samples were immunoprecipitated (IP) by FLAG and immunoblotted (IB) by Myc. E and F, physical interaction between Gli1/2, Runx2, and Smad1 was assessed by co-IP in which the samples were immunoprecipitated by FLAG and immunoblotted by Myc. Simultaneous interaction of Gli1/Runx2/Smad1 or Gli2/Runx2/Smad1 was seen in the last lane of E or F. G, CHIP on COL10A1 promoter was performed using HTB94 human chondrosarcoma cells. Sonicated and purified chromatin was incubated with antibodies as shown in the figure. Endogenous binding of Gli, Runx2, and pSmads was detected in the COL10A1 basic promoter region. An α-histone antibody was used as a positive control, and rabbit or mouse IgGs were used as a negative control. The detection was confirmed in three independent experiments. The representative data are shown here.

FIGURE 8.

Ihh induces matrix mineralization in primary chondrocytes interacting with BMP/Runx2/Smads pathway. A–D, Ihh was overexpressed in primary chondrocytes with or without treatment by additional reagents (A, B, and D) or viruses (C) as shown in the figures, and an in vitro mineralization assay was performed under stimulation by β-glycerophosphate and assessed by Alizarin red staining (n = 3 or 4). E, an in vitro mineralization assay was performed in the presence with Runx2 overexpression with or without cyclopamine (n = 3 or 4). F, diagram showing how Ihh downstream signaling molecules Gli1/2 together with Runx2/Smads regulate transcription and expression of Col X.