Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway

Abstract

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

Keywords: igamentum flavum; cyclic stretch; ossification; osteogenic differentiation; pathway; Indian hedgehog.

Figure 1.

Expression of RUNX2, Osterix, ALP and OCN in LF and OLF tissues. Reverse transcription-quantitative PCR results for the mRNA expression levels of (A) RUNX2, (B) Osterix, (C) ALP and (D) OCN in the LF and OLF groups. Immunohistochemistry images of (E) RUNX2, (F) Osterix, (G) ALP and (H) OCN. Quantification of the protein expression levels of (I) RUNX2, (J) Osterix, (K) ALP and (L) OCN in the LF group and OLF group. Magnification, ×400. The red arrows represent the typical cells, which were magnified and shown in the upper right-hand corner (magnification, × 1,600). N=8. **P<0.01, ***P<0.001 vs. LF samples. OLF, ossification of ligamentum flavum; LF, ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin.

Figure 2.

Expression level of IHH in LF and OLF tissues. (A) Reverse transcription-quantitative PCR results showed the mRNA expression levels of IHH between the LF group and OLF group. (B) Representative images of immunohistochemistry staining from each group. Magnification, ×400. The red arrows represent the typical cells, which were magnified and shown in the upper right-hand corner (magnification, ×1,600). (C) Immunohistochemistry results of the protein expression levels of IHH in the LF group and OLF group. N=8. **P<0.01 vs. LF samples. IHH, Indian hedgehog; OLF, ossification of ligamentum flavum; LF, ligamentum flavum.

Figure 3.

Expression level of osteogenic genes and IHH signaling genes in LF and OLF cells after cyclic stretch. (A) Reverse transcription-quantitative PCR results showed that in LF cells, cyclic stretch increased the ALP expression level, while other gene expression levels did not change. (B) Relative mRNA expression levels of RUNX2, Osterix, ALP, OCN, IHH and SMO significantly increased after the application of cyclic stretch in OLF cells. *P<0.05, **P<0.01 vs. 0 h. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; LF, ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; IHH, Indian hedgehog.

Figure 4.

Effect of cyclic stretch on osteogenic genes and ALP activity. Expression levels of (A) RUNX2, (B) Osterix, (C) ALP and (D) OCN in OLF cells were detected after cyclic stretch treatment for 0, 6, 12 and 24 h. Protein expression levels of (E) RUNX2, (F) Osterix, (G) ALP and (H) OCN after cyclic stretch. (I) Representative images of ALP staining. Magnification, ×400. (J) Cyclic stretch increased ALP activity in OLF cells. *P<0.05, **P<0.01, ***P<0.001. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin.

Figure 5.

Effect of cyclic stretch on the expression of molecules involved in the IHH signaling pathway in OLF cells. Cyclic stretch of OLF cells resulted in significant increases in the (A) mRNA and (B) protein expression levels of IHH. Cyclic stretch of OLF cells resulted in significant increases in the (C) mRNA and (D) protein expression levels of SMO at 6, 12 and 24 h. mRNA expression levels of (E) GLI1 and (F) GLI2 were significantly increased after cyclic stretch. (G) No significant effect was observed on the expression level of GLI3. *P<0.05, **P<0.01, ***P<0.001. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; IHH, Indian hedgehog; SMO, Smoothened; GLI, GLI family zinc finger.

Figure 6.

Effect of the Indian hedgehog signaling pathway inhibitor Cpn on the expression levels of RUNX2, Osterix, ALP and OCN in OLF cells. mRNA expression levels of (A) RUNX2, (B) Osterix, (C) ALP and (D) OCN. Protein expression levels of (E) RUNX2, (F) Osterix, (G) ALP (H) and OCN were significantly decreased in the stretch + Cpn group compared with cyclic stretch alone. (I) Representative images of ALP staining. Magnification, ×400. (J) ALP activity was significantly reduced in the Cpn + stretch group compared with cyclic stretch alone. *P<0.05, **P<0.01, ***P<0.001. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; RUNX2, Runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; Cpn, cyclopamine.

Figure 7.

Effect of Cpn on the expression levels of Indian hedgehog signaling-related genes in OLF cells. mRNA expression levels of (A) SMO, (B) GLI1, (C) GLI2 and (D) GLI3. *P<0.05, ***P<0.001. Data are presented as the mean ± SD. N=3. OLF, ossification of ligamentum flavum; Cpn, cyclopamine; ns, not significant; SMO, Smoothened; GLI, GLI family zinc finger.