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.
Highlights
Bone remodeling is coordinated by the balance between bone formation and resorption.Imbalance of the bone formation and resorption causes metabolic bone diseases, including osteoporosis
To investigate the role of 4.1G in bone formation, we used the tibia of a newborn mouse and a mouse preosteoblast cell line, MC3T3-E1, and found that 4.1G is essential for the primary ciliogenesis, the ciliary Hh-signaling, osteoblast differentiation, and the resulting bone formation in the newborn tibia
The reduction was especially prominent in the trabecular bone of the 4.1G-KO tibia, indicating that 4.1G regulates trabecular bone formation in preosteoblasts and osteoblasts (Figure S1)
Summary
Bone remodeling is coordinated by the balance between bone formation and resorption. Imbalance of the bone formation and resorption causes metabolic bone diseases, including osteoporosis. The cilium regulates cell differentiation and proliferation in various types of cells by mediating signals through selective membrane receptors distributed on its membrane surface, including G protein-coupled receptors (GPCRs), growth factor receptors, and ion channels [5–7]. Among these, smoothened, a cilia localizing GPCR, upregulates genes for the transcription factor Gli and Hedgehog (Hh) receptor patched 1 (Ptch1) and is required for bone formation by promoting osteoblast differentiation [8,9]. Knockout of cilia formation-associated molecules, including intraflagellar transport (IFT) proteins and kinesins, causes bone malformation, demonstrating the importance of the primary cilia in preosteoblasts for bone formation. To investigate the role of 4.1G in bone formation, we used the tibia of a newborn mouse and a mouse preosteoblast cell line, MC3T3-E1, and found that 4.1G is essential for the primary ciliogenesis, the ciliary Hh-signaling, osteoblast differentiation, and the resulting bone formation in the newborn tibia
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