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. 2012 Sep;51(3):407-17.
doi: 10.1016/j.bone.2012.06.021. Epub 2012 Jul 4.

The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

Shuying Yang  1 , Changdong Wang
Affiliations

The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

Shuying Yang et al. Bone. 2012 Sep.

Abstract

Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia, which play important roles in development and homeostasis. IFT80 is a newly defined IFT protein. Partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development. However, the role and mechanism of IFT80 in osteogenesis is unknown. Here, we first detected IFT80 expression pattern and found that IFT80 was highly expressed in mouse long bone, skull, and during osteoblast differentiation. By using lentivirus-mediated RNA interference (RNAi) technology to silence IFT80 in murine mesenchymal progenitor cell line-C3H10T1/2 and bone marrow derived stromal cells, we found that silencing IFT80 led to either shortening or loss of cilia and the decrease of Arl13b expression - a small GTPase that is localized in cilia. Additionally, silencing IFT80 blocked the expression of osteoblast markers and significantly inhibited ALP activity and cell mineralization. We further found that IFT80 silencing inhibited the expression of Gli2, a critical transcriptional factor in the hedgehog signaling pathway. Overexpression of Gli2 rescued the deficiency of osteoblast differentiation from IFT80-silenced cells, and dramatically promoted osteoblast differentiation. Moreover, introduction of Smo agonist (SAG) promotes osteoblast differentiation, which was partially inhibited by IFT80 silencing. Thus, these results suggested that IFT80 plays an important role in osteogenesis through regulating Hedgehog/Gli signal pathways.

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Figures

Fig. 1
Fig. 1
IFT80 is prominently expressed in bone tissue and during osteoblast differentiation. (A) Real time RT-PCR analysis of IFT80 expression in mouse tissues. Total RNA was extracted from mouse tissues: brain, liver, eye, lung, heart, kidney, spleen, muscle, long bone and skull. (B) Immunostaining for IFT80 expression. Bright green is positive for expression of IFT80 (red arrows). (C) Real time RT-PCR analysis for IFT80 at transcriptional level during osteoblast and osteoclast differentiation. C3H10T1/2 cells and BMSCs were respectively induced with OS media for 0, 7 14 and 21 days respectively. Raw264.7 cells and BMMs were respectively induced with M-CSF/RANKL (20ng/ml) for 0, 1, 2 and 4 days. (D) Western Blot analysis of IFT80 protein expression during osteoblast differentiation. C3H10T1/2 cells were induced with OS media for 0, 3, 7, 14 and 17 days. (E) Quantification of protein levels from immunblots as in D. The protein levels of IFT80 were normalized to GAPDH.
Fig. 2
Fig. 2
Silencing IFT80 inhibits the expression of Arl13b, IFT80, gamma tubulin and acetylated α-tubulin, and impairs cilia formation. (A) Western blot analysis of IFT80 expression. C3H10T1/2 cells were infected with pGIPZ-IFT80 shRNA (I1–I5) or pGIPZ-scrambled shRNA (PGIPZ) lentiviruses for 48 hrs, and then the protein was extracted to detect IFT80 expression. IFT80 expression was silenced in I1 and I3. (B) Quantitative analysis of protein levels from immunoblots as in A. The protein levels of IFT80 were normalized to GAPDH. (C) Immunofluorescence staining for IFT80 location and expression. IFT80 was expressed in control group (pGIPZ, red), but blocked in IFT80-silenced cells. Nuclear were stained with DAPI (blue). (D) Immunofluorescence staining for Arl13b location and expression. Arl13b was expressed in control group (pGIPZ, red)), but not in IFT80-silenced cells. Nuclear were stained with DAPI (blue). (E) Immunofluorescence staining for cilia by detecting the location and expression of gamma tubulin (red) and acetylated α-tubulin (bright blue). Nuclear were stained with DAPI (dark blue). Cilia existed in control cells, but not in IFT80-silenced cells.
Fig. 3
Fig. 3
Silencing IFT80 inhibits osteoblastic ALP activity, osteoblast marker gene expression, and cell mineralization. (A) ALP activity assay. C3H10T1/2 cells were infected with pGIPZ-IFT80 shRNA (I3) or pGIPZ-scrambled shRNA (pGIPZ) lentiviruses, induced with OS media for 7 days, and then ALP activity was examined. ALP activity in control group was 2.9 fold of that in IFT80-silenced group (n=6, *p<0.01). (B) Real time RT-PCR for analyzing the expression of RUNX2, OCN, BSP and ALP. C3H10T1/2 cells were infected with pGIPZ-IFT80 shRNA (I3) or pGIPZ-scrambled shRNA (pGIPZ) lentiviruses and induced with OS media for 7 days. (C) Von Kossa and Alizarin Red staining. C3H10T1/2 cells were infected with I3 or pGIPZ as described in Material and Methods, and then induced with OS media for 14 days. (D) Quantitative mineralization level based on Alizarin Red staining. The mineralized level in IFT80-silenced cells was significantly lower than that in control cells (n=6, *p<0.05). (E–G) Silence of IFT80 in mouse BMSCs. Primary BMSCs, derived from mouse bone marrow, were infected with I3 or pGIPZ and then induced with OS media for: E, 2 days for western blotting; F, 14 days for Alizarin Red staining; G, 7 days for ALP assays (n=6, *p<0.05).
Fig. 4
Fig. 4
Ectopic expression of Gli2 rescues the impaired osteoblast differentiation and mineralization resulting from IFT80 silencing. (A) Western blot analysis of Gli2 expression. C3H10T1/2 cells were infected with pGIPZ-shRNA IFT80 (I3) or pGIPZ-scrambled shRNA (pGIPZ) lentiviruses for 48 hrs, at which time the protein was extracted for detecting Gli2 expression. (B) Quantitative analysis of protein levels from immunoblots as in a. The protein levels of Gli2 were normalized to GAPDH. (C) Western blot analysis for ectopic expression of Gli2. C3H10T1/2 cells were infected with the Gli2-expressing retrovirus (Gli2) or with a control retrovirus (pBMN). Gli2 protein level was significantly increased in Gli2-overexpresed cells compared with the control cells. (D) Quantitative analysis of protein levels from immunoblots as in c. The protein levels of Gli2 were normalized to GAPDH. (E–F) ALP activity assay. C3H10T1/2cells were first infected with either I3 or pGIPZ for 48hrs, followed by infection with pBMN or Gli2 for an additional 48 hours. The cells were then induced with OS media for 7 days before detection of the ALP activity assay. Each bar is expressed as the mean ± SD (unit/min*mg DNA) of six determinations: Gli2 vs. I3, pGIPZ, or I3+Gli2, ***p30.001; I3 vs. I3+Gli2 or pGIPZ, *p30.001; I3+Gli2 vs. pGIPZ, **p>0.05. (f) Von Kossa and Alizarin Red staining. After the infection protocol outlined above in e, cells were induced in OS media for 14 days prior to Von Kossa and Alizarin Red staining. (G) Quantitative mineralization level based on Alizarin Red staining. Each bar is expressed as the mean ± SD (unit/min*mg DNA) of three determinations. Gli2 vs. I3, pGIPZ, or I3+Gli2, ***p30.001; I3 vs. I3+Gli2 or pGIPZ, *p30.001; I3+Gli2 vs. pGIPZ, **p>0.05.
Fig. 5
Fig. 5
IFT80/Gli2 regulates osteoblast marker gene expression. C3H10T1/2 cells were first infected with either I3 or pGIPZ for 48hrs, followed by infection with pBMN or Gli2 for an additional 48 hours, and then induced with OS media for 7 days. (A) RT-PCR. Overexpression of Gli2 significantly increased BMP2, ALP and RUNX2 expression compared with that in the control cells. (B) Real time RT-PCR. Overexpression of Gli2 increased by 2.7, 2.8 and 3.9 fold the expression of BMP2, ALP and RUNX2, respectively. (C) Western Blot. The expression of Col I and OCN was attenuated in IFT80-silenced cells, but rescued and increased in Gli2- overexpressed cells. (D) Quantitative analysis of protein levels from immunoblots as in C. The protein levels were normalized to GAPDH.
Fig. 6
Fig. 6
IFT80 silencing inhibits the SAG induced osteoblast differentiation and mineralization. (A–B) C3H10T1/2 cells were infected with pGIPZ-IFT80 shRNA (I3) or pGIPZ-scrambled shRNA (pGIPZ) lentiviruses, and treated with OS media with 10nM SAG for 24 days to study effects on cell mineralization. (A) Alizarin Red staining. Quantitative mineralization level based on Alizarin Red staining reported as the mean ± SD from tests of samples in triplicate. There are significant differences between the SAG group vs. other groups (**p<0.01), and between I3 vs. pGIPZ or I3+SAG (***P<0.001). There is no different between pGIPZ vs. I3+SAG (* P>0.05), indicating that SAG completely rescued the deficiency of osteoblast mineralization resulting from silencing IFT80. (B) Von kossa staining. (C–D) Western blots. C3H10T1/2 cells were infected with I3 or pGIPZ, followed by co-infection with Gli2-expressing retrovirus (Gli2) or with a control retrovirus (pBMN) for 24 hrs. The infected cells were treated with OS media with or without 10nM SAG for 7 days for analysis of Gli2 expression. Overexpression of Gli2, or treatment with SAG, up-regulates the expression of Gli2 in IFT80-silenced cells as well as control cells.
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