Four and Half Lim Protein 2 (FHL2) Stimulates Osteoblast Differentiation

Abstract

FHL2, a molecule that interacts with many integrins and transcription factors, was found to play an important role in osteoblast differentiation. Overexpression of FHL2 increases the accumulation of osteoblast differentiation markers and matrix mineralization, whereas FHL2 deficiency results in inhibition of osteoblast differentiation and decreased bone formation.Introduction: Integrin-matrix interaction plays a critical role in osteoblast function. It has been shown that the cytoplasmic domains of integrin ␤ subunits mediate signal transduction induced by integrin-matrix interaction. We reasoned that the identification of proteins interacting with ␤-cytoplasmic tails followed by analysis of the function of these proteins would enhance our understanding on integrin signaling and the roles of these proteins in osteoblast activities. Materials and Methods: Yeast two hybrid assay was used to identify proteins interacting with the cytoplasmic domain of integrin ␤5 subunit. The association of these proteins with integrin ␣v␤5 was confirmed by confocal analysis and co-immunoprecipitation. A stable MC3T3-E1 cells line overexpressing Four and Half Lim Protein 2 (FHL2) and mouse osteoblasts deficient in FHL2 were used to study the roles of FHL2 in osteoblast differentiation and bone formation. Matrix protein expression was determined by mRNA analysis and Western blotting. Matrix mineralization was detected by Alizarin red staining. Alkaline phosphatase activity was also measured. CT was used to determine bone histomorphometry. Results and Conclusions: FHL2 and actin-binding proteins, palladin and filamin A, were identified as proteins interacting with ␤5 cytoplasmic domain. FHL2 co-localized with ␣v␤5 at the focal adhesion sites in association with palladin and filamin A. FHL2 was also present in nuclei. Osteoblasts overexpressing FHL2 exhibited increased adhesion to and migration on matrix proteins. Conversely, FHL2 stimulation of CREB activity was dependent on integrin function because it was inhibited by Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The expression of osteoblast differentiation markers and Msx2 was upregulated, and bone matrix mineralization was increased in FHL2 overexpressing cells. In contrast, FHL2-deficient bone marrow cells and osteoblasts displayed decreased osteoblast colony formation and differentiation, respectively, compared with wildtype cells. Moreover, FHL2-deficient female mice exhibited greater bone loss than the wildtype littermates after ovariectomy. Thus, FHL2 plays an important role in osteoblast differentiation and bone formation.

Four-and-a-Half LIM Domain Protein 2 Is a Novel Regulator of Sphingosine 1-Phosphate Receptor 1 in CCL19-Induced Dendritic Cell Migration

“…The question remains how FHL2 regulates DC migration and S1PR1 expression under physiological circumstances. In fibroblasts, we showed that S1P signaling causes RhoA-and Rockdependent translocation of FHL2 into the nucleus (21), where it can function as coactivator or corepressor for several transcription factors (22)(23)(24)(25). This is most likely due to S1PR2-mediated activation of RhoA and Rock (18) (Fig.…”

“…We previously showed that S1P triggers a signaltransduction cascade mediating nuclear translocation of the LIMonly protein FHL2 in response to activation of the RhoA GTPase (20,21). Following nuclear translocation, FHL2 can function as a transcriptional coactivator for, among others, the androgen receptor (20), CREB (22), AP-1, RunX1 (23), and serum response factor (24), linking extracellular signals to gene expression (25). We previously showed that FHL2-deficient mice have a severely delayed wound healing response due a defect in fibroblast migration and a reduced collagen contraction (26,27).…”

“…Directional migratory speed in DCs is known to be regulated by CCR7, among others (7). Because FHL2 can act as a transcriptional coactivator or corepressor (20)(21)(22)(23)29) and, thus, might affect CCR7 expression, we investigated whether CCR7 expression was increased in FHL2 BMDCs also had altered migratory properties in vivo. To this end, FIGURE 1.…”

Female mice lacking p47phoxhave altered adipose tissue gene expression and are protected against high fat-induced obesity

“…9). In addition, other pro-osteoblastic genes, Ihh (Indian hedgehog) (10) and four-and-a half LIM 2 (Fhl2) (9,16), were elevated in the P47KO HFD group compared with the WT:HFD group. Gja1 (gap junction protein alpha 1, connexin 36), another regulator of osteoblast development (3,22), was also elevated in the P47KO HFD group (Fig.…”

Realizing the potential of gene-based molecular therapies in bone repair

“…Four and half lim protein-2 (FHL2) and Lim mineralization proteins 1 and 3 (LMP 1 and LMP3) are recently investigated proteins that were shown to induce bone regeneration. (144,145) LMP-1 and LMP-3 can also act synergistically with BMPs. (146,147) NEL-like molecule-1 (Nell-1) is another molecule under investigation for its osteogenic properties in repair of large segmental defects.…”