Focal adhesion kinase activity is required for bone morphogenetic protein--Smad1 signaling and osteoblastic differentiation in murine MC3T3-E1 cells.
Cell-matrix interactions via integrins are essential for osteoblastic differentiation. We have shown that signals activated by aggregation of alpha2beta1-integrin with type I collagen are involved in the differentiation of osteoblastic MC3T3-E1 cells. Focal adhesion kinase (FAK) is an immediate downstream signal of the beta1-integrin, and inactivation of FAK has been shown to disrupt osteoblastic differentiation. To elucidate roles of FAK in osteoblastic cells, we examined MC3T3-E1 cells stably expressing antisense FAK (asFAK) messenger RNA (mRNA). Alkaline phosphatase (ALP) activity, an osteoblastic marker, did not increase in asFAK cells with a long-term culture until 21 days or in response to bone morphogenetic protein 2 (BMP-2). Treatment with BMP-2 also failed to stimulate the expression of osteocalcin in asFAK cells. In control MC3T3-E1 cells, BMP-2 induced translocation of Smad1 into nuclei to stimulate transcriptional activity of the Smad6 promoter gene that contains a Smad1 response element. In contrast, BMP-2 failed to increase transcriptional activity of Smad6 promoter gene in asFAK cells, although it induced nuclear translocation of Smad1. These results indicate that FAK was involved in Smad1-dependent transcriptional activity but not in nuclear translocation of Smad1 in osteoblastic cells. Hence, FAK activation by integrins might converge transcriptional activation by BMP of its target genes in osteoblastic cells. These observations suggest that FAK activity is essential for BMP-Smad signaling to stimulate osteoblastic differentiation.
Tamura
Y
Y
Department of Medicine, University of Tokyo School of Medicine, Japan.
Takeuchi
Y
Y
Suzawa
M
M
Fukumoto
S
S
Kato
M
M
Miyazono
K
K
Fujita
T
T
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism* / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins; D051898:Smad1 Protein; D051901:Smad4 Protein; D051902:Smad5 Protein; D051905:Smad6 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta
D000199:Actins; C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; C497399:SMAD1 protein, human; C497796:SMAD4 protein, human; C497797:SMAD5 protein, human; C497802:SMAD6 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D051902:Smad5 Protein; C497798:Smad5 protein, mouse; D051905:Smad6 Protein; C497803:Smad6 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse