Abstract
Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of alphabeta(1), alpha(v)beta(3), and alpha(v)beta(5) integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.
Highlights
For bone formation to occur, osteoblast precursor cells must migrate from bone marrow compartment to bone surface where they differentiate and deposit bone matrix
Generation of Human Osteoblastic Cell Lines Expressing a Dominant Negative Erk1 Protein—Since Extracellular signal-regulated kinases (Erks) mediate both growth factor- and integrin- induced signal transduction, it is likely that they play important role in the regulation of osteoblast function
We have demonstrated that the expression of an Erk1 dominant negative protein inhibited Erk/MAPK activity, resulting in decreased proliferation and differentiation of human osteoblastic cells
Summary
Materials—Type I collagen (Vitrogen 100) was from Cohesion (Palo Alto, CA). Recombinant osteopontin was purified as described previously [33]. [125I]NaI and Tran35S-Label were from ICN (Costa Mesa, CA). [3H]Thymidine, [␥-32P]ATP, ECL kit, and protein G-Sepharose were from Amersham Pharmacia Biotech. AP-1 consensus oligonucleotide (5Ј-CGCTTGATGAGTCAGCCGGAA-3Ј) and T4 polynucleotide kikinase; FAK, focal adhesion kinase; EMSA, electrophoretic mobility shift assay
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