Cell-matrix interaction in osteoblastic differentiation and its disorder in involutional osteoporosis

1)成骨细胞分化中胶原-整合素相互作用激活的细胞内信号我们之前已经表明，I型胶原（COL）与α 2 β 1整合素的相互作用导致成骨细胞分化。在本研究中，为了阐明细胞-基质相互作用如何调节这些表型变化，我们在小鼠MC3T3-E1细胞中检测了信号通路。细胞附着于COL刺激了局灶黏附激酶（FAK）和丝裂原活化蛋白激酶（MAPK）的酪氨酸磷酸化，并增强了MAPK的活性。酪氨酸激酶的抑制、局灶黏附的破坏或反义FAK mRNA的过表达均可阻止MAPK的激活和碱性磷酸酶（ALP）活性的增加。mapk特异性磷酸酶的瞬时表达也抑制了ALP活性的升高。此外，在没有胶原蛋白产生的情况下，引入具有组成活性的MAPK激酶可增强ALP活性。这些结果表明，COL-alpha2beta1整合素相互作用通过激活FAK及其下游信号促进分化。这些信号通路可能在成骨细胞的序贯分化中起重要作用。2)基质相关bmp在成骨细胞分化中的重要作用成骨细胞谱系中细胞向成熟成骨细胞分化的内在机制尚不清楚。骨形态发生蛋白（BMPs）刺激成骨细胞分化和骨形成。我们证明MC3T3-E1细胞组成性地表达BMP-2和BMP-4的mrna，并在富含胶原的细胞外基质中积累bmp。与细胞外基质相关的bmp参与了成骨细胞分化的诱导。MC3T3-E1细胞组成性表达IA型和II型BMP受体。当将一种激酶缺陷型IA型BMP受体稳定转染到MC3T3-E1细胞中以消除BMP-2/4信号时，这些细胞不仅对外源性BMP-2没有反应，而且失去了向成骨细胞分化的能力。这些观察结果表明，积累在细胞外基质中的内源性BMP-2/4对于成骨细胞谱系中细胞的成骨分化至关重要。因此，BMP-2/4在成骨细胞中的调控机制是研究骨质疏松发病机制的首要问题。晚期糖基化终产物（AGE）随着年龄的增长在基质中积累。胶原蛋白最容易发生广泛的糖基化，AGE被认为与几种疾病有关。AGE抑制细胞内信号传导，如酪氨酸磷酸化，由附着于成骨细胞的胶原基质引起。因此，年龄诱导的成骨细胞损伤在绝经性骨质疏松的发病机制中有待进一步研究。少

1) Intracellular signaling activated by collagen-integrin interactions in osteoblastic differentiationWe have previously shown that interaction of type I collagen (COL) with alpha2beta1 integrin causes differentiation in osteoblastic cells. In this study, to clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase, destruction of focal adhesion, or overexpression of antisense FAK mRNA prevented the activation of MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase also suppressed the elevation of ALP activity. Furthermore, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. These results demons … More trate that COL-alpha2beta1 integrin interaction facilitates differentiation via the activation of FAK and its downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts.2) Essential role of matrix-associated BMPs in osteoblastic differentiationIntrinsic mechanisms whereby cells in an osteoblast lineage differentiate into mature osteoblasts are yet unclear. Bone morphogenetic proteins (BMPs) stimulate osteoblastic differentiation and bone formation. We demonstrate that MC3T3-E1 cells constitutively expressed mRNAs for BMP-2 and BMP-4 and accumulated BMPs in collagen-rich extracellular matrices. BMPs associated with the extracellular matrices were involved in the induction of osteoblastic differentiation. MC3T3-E1 cells constitutively expressed type IA and type II BMP receptors. When a kinase-deficient type IA BMP receptor was stably transfected to MC3T3-E1 cells to obliterate BMP-2/4 signaling, these cells not only failed to respond to exogenous BMP-2 but lost their capability of differentiation into osteoblasts. These observations suggest that endogenous BMP-2/4 accumulated in extracellular matrices are essential for the osteoblastic differentiation of cells in the osteoblast lineage. Therefore, the regulatory mechanism of BMP-2/4 actions in osteoblastic cells is a principal issue to be elucidated for better understanding of pathogenesis of osteoporosis.3) Advanced glycation endproducts impair cell-collagen interactions in osteoblastic cellsAdvanced glycation endproducts (AGE) accumulate in matrix with advancing age. Collagen is most susceptible to extensive glycation, and AGE is suggested to be involved in several diseases. AGE inhibited intracellular signaling, such as tyrosine phosphorylation, evoked by the attachment to collagen matrix in osteoblastic cells. Thus, AGE-induced impairment of osteoblastic cells are further to be investigated for the pathogenesis of involutional osteoporosis. Less