Cytoskeletal Protein 4.1G Is Essential for the Primary Ciliogenesis and Osteoblast Differentiation in Bone Formation

Abstract

The primary cilium is a hair-like immotile organelle with specific membrane receptors, including the receptor of Hedgehog signaling, smoothened. The cilium organized in preosteoblasts promotes differentiation of the cells into osteoblasts (osteoblast differentiation) by mediating Hedgehog signaling to achieve bone formation. Notably, 4.1G is a plasma membrane-associated cytoskeletal protein that plays essential roles in various tissues, including the peripheral nervous system, testis, and retina. However, its function in the bone remains unexplored. In this study, we identified 4.1G expression in the bone. We found that, in the 4.1G-knockout mice, calcium deposits and primary cilium formation were suppressed in the trabecular bone, which is preosteoblast-rich region of the newborn tibia, indicating that 4.1G is a prerequisite for osteoblast differentiation by organizing the primary cilia in preosteoblasts. Next, we found that the primary cilium was elongated in the differentiating mouse preosteoblast cell line MC3T3-E1, whereas the knockdown of 4.1G suppressed its elongation. Moreover, 4.1G-knockdown suppressed the induction of the cilia-mediated Hedgehog signaling and subsequent osteoblast differentiation. These results demonstrate a new regulatory mechanism of 4.1G in bone formation that promotes the primary ciliogenesis in the differentiating preosteoblasts and induction of cilia-mediated osteoblast differentiation, resulting in bone formation at the newborn stage.

Keywords: bone formation; cytoskeletal protein 4.1G; preosteoblasts; primary cilium.

Figure 1

Protein 4.1G regulates osteogenesis of the newborn tibia. (A,B) Expression levels of 4.1G in wild-type (WT) and 4.1G-knockout (KO) femur. Femurs were isolated from the 4-week-old male (A) and female (B) mice. Expression levels of 4.1G were analyzed by Western blotting. Cell lysates were immunoblotted (IB) with the anti-4.1G or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. (C–F) Calcium deposit. Newborn tibia sections from the male (C,E) and female (D,F) mice were stained by modified von Kossa’s reaction. (E,F) The area of calcium deposit was calculated as %WT of tibia. (G–N) Tibia morphology. Newborn tibia sections from the male (G,K,M) and female (H,J,L,N) mice were stained with hematoxylin and eosin. Black bar, whole tibia; blue bar, proliferating zone; green bar, hypertrophic zone; red bar, bone tissue. The length of the whole tibia (I,J), proliferating and hypertrophic zones (K,L), and bone tissue (M,N). Data are presented as the mean ± standard deviation (S.D.) from 4 ((E); WT), 6 ((E); KO), 7 ((F); WT), 6 ((F); KO), 4 ((I,K,M); WT), 6 ((I,K,M); KO), 5 ((J,L,N); WT), and 5 ((J,L,N); KO) independent experiments. # p < 0.05, Student’s t-test. n.s., not significant. Scale bars, 500 µm.

Figure 2

Protein expression levels of 4.1G decrease during the differentiation of MC3T3-E1 cells. The confluent MC3T3-E1 cells were treated with (triangle) or without (circle) 250 µM ascorbic acid and 50 µM β-glycerol phosphate (AA/βGP) for up to 20 days. (A) Alkaline phosphatase (ALP) activity in the whole cell lysates was measured at the indicated time points. The activity was normalized by the protein content. (B) Calcium deposit on the cell surface was quantified by Alizarin red staining. The intensity of calcium deposit on the AA/βGP-treated cells of day 20 was considered as 100%. (C) Protein content in the whole cell lysate was measured at the indicated time points. (D) Protein expression levels of 4.1G were analyzed by Western blotting. Whole cell lysates were immunoblotted (IB) with the anti-4.1G or anti-GAPDH antibody. One of four independent representative immunoblots is shown. (E) Relative 4.1G expression levels of (D). The amount of 4.1G protein expression at day 0 was considered as 100%. Data are presented as the mean ± standard error of the mean (S.E.M.) from 3 (A–C) and 4 (E) independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, two-way analysis of variance (ANOVA) followed by Bonferroni’s test (vs. Control of each day).

Figure 3

Primary cilia are elongated in the differentiating MC3T3-E1 cells. Confluent MC3T3-E1 cells were treated in the presence or absence of AA/βGP for 4, 10, and 20 days. (A) The cells were immunolabeled with an anti-acetylated α-tubulin (Ac-Tub) antibody. The cell nuclei were labeled by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). The boxed areas indicate the enlarged regions. (B) Quantification of the cilia length. Data are presented as the mean ± S.E.M. from 29–87 cilia. * p < 0.05, *** p < 0.001, two-way ANOVA followed by Bonferroni’s test (vs. Control of each day). n.s., not significant. Scale bar, 20 µm.

Figure 4

Protein 4.1G promotes ciliogenesis in preosteoblasts. (A–F) Ciliogenesis at the trabecular bone in newborn tibia. Newborn tibiae were isolated from the male (A,C,E) and female (B,D,F) WT or 4.1G-KO mice. (A,B) The cilia were immunolabeled with an anti-Arl13b antibody. The cell nuclei were labeled by DAPI. The boxed areas indicate the enlarged regions. (C–F) Percentages of ciliated cells (C,D) and cilia length (E,F) at the trabecular bone are shown. (G,H) Ciliogenesis in MC3T3-E1 cells. (G) MC3T3-E1 cells transfected with the control, 4.1G-sh1, or 4.1G-sh2 were treated with AA/βGP for 2 and 4 days, respectively. Representative confocal microscope images of primary cilia (Ac-Tub) in control- or 4.1G-sh1-transfected MC3T3-E1 cells. (H) Percentages of primary cilia-positive cells are shown. (C–F) Data are presented as the mean ± S.D. from 4 ((C); WT), 6 ((C); KO), 5 ((D); WT), and 5 ((D); KO) independent experiments. In the cilia length experiment, 285–380 cilia were measured (E,F). ^# p < 0.05, Student’s t-test; n.s., not significant. (H) Data are presented as the means ± S.E.M. from three independent experiments. ^§§ p < 0.01, ^§§§ p < 0.001, one-way ANOVA followed by Tukey’s test (vs. Day 0 of the group). ** p < 0.01, *** p < 0.001, two-way ANOVA followed by Bonferroni’s test. Scale bars, (A,B) 50 µm (wide view) and 10 µm (enlarged view), (G) 20 µm.

Figure 5

Protein 4.1G increases primary cilia-mediated osteogenic signals. MC3T3-E1 cells transfected with the control or 4.1G-sh1 were treated with AA/βGP for 2 days. The cells were further stimulated with 2 µM of purmorphamine for indicated time periods. Relative mRNA expression levels of Gli1 (A), patched 1 (Ptch1) (B), osterix (C), or osteocalcin (D) were normalized to GAPDH level. Data are presented as the mean ± S.E.M. from three independent experiments. ^§ p < 0.05, ^§§ p < 0.01, ^§§§ p < 0.001, one-way ANOVA followed by Tukey’s test (vs. 0 h of the group). * p < 0.05, *** p < 0.001, two-way ANOVA followed by Bonferroni’s test.

Figure 6

Protein 4.1G supports osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 cells transfected with the control or 4.1G-sh1 were treated with AA/βGP for 2 and 4 days. (A) Alkaline phosphatase (ALP) activity in the cell lysate was measured. The activity was normalized by the protein content. (B) Protein content in the whole cell lysate was measured at the indicated time points. Data are presented as the mean ± S.E.M. from five independent experiments. ^§ p < 0.05, one-way ANOVA followed by Tukey’s test (vs. Day 0 of the group). *** p < 0.001, two-way ANOVA followed by Bonferroni’s test. n.s., not significant.