Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions

Abstract

The reciprocal and adaptive interactions between cells and substrates governing morphological transitions in the osteoblast compartment remain largely obscure. Here we show that osteoblast cultured in basement membrane matrix (Matrigel™) exhibits significant morphological changes after ten days of culture, and we decided to exploit this situation to investigate the molecular mechanisms responsible for guiding osteoblast morphological transitions. As almost all aspects of cellular physiology are under control of kinases, we generated more or less comprehensive cellular kinome profiles employing PepChip peptide arrays that contain over 1000 consensus substrates of kinase peptide. The results obtained were used to construct interactomes, and these revealed an important role for FoxO in mediating morphological changes of osteoblast, which was validated by Western blot technology when FoxO was significantly up-expressed in response to Matrigel™. As FoxO is a critical protein in canonical hedgehog signalling, we decided to explore the possible involvement of hedgehog signalling during osteoblast morphological changes. It appeared that osteoblast culture in Matrigel™ stimulates release of a substantial amounts Shh while concomitantly inducing upregulation of the expression of the bona fide hedgehog target genes Gli-1 and Patched. Functional confirmation of the relevance of these results for osteoblast morphological transitions came from experiments in which Shh hedgehog signalling was inhibited using the well-established pathway inhibitor cyclopamine (Cyc). In the presence of Cyc, culture of osteoblasts in Matrigel™ is not capable of inducing morphological changes but appears to provoke a proliferative response as evident from the upregulation of Cyclin D3 and cdk4. The most straightforward interpretation of our results is that hedgehog signalling is both necessary and sufficient for membrane matrix-based morphological transitions.

Nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as prerequisite to ECM remodeling

“…Since osteoblast differentiation requires Src inhibition and nHA promotes osteoblast differentiation [17], we suggested the up-expression of the Src was due to compensation feedback, as reported earlier for Src during osteoblast adhesion (up to 24 h) and MMPs. Importantly, we demonstrated that Src is necessary to govern cytoskeletal remodeling during osteoblast morphological changes [29].…”

“…Then, membranes were blocked with either 1% fat-free dried milk or bovine serum albumen (2.5%) in 1× Tris-buffered saline (TBS)-Tween 20 (0.05%, vol/vol) and incubated overnight at 4 • C with appropriate primary antibody at 1:1000 dilutions. After washing in 3× TBS-Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgGs antibodies, at 1:2000 dilutions (in all immunoblotting assays), in blocking buffer for 1 h. After washing in 3 x TBS, the detection was performed by using enhanced chemiluminescence (ECL) [29].…”

Mg–Al and Zn–Al Layered Double Hydroxides Promote Dynamic Expression of Marker Genes in Osteogenic Differentiation by Modulating Mitogen‐Activated Protein Kinases

“…Specific to bone, the therapeutic biomaterial should have osteopromotive characteristics, which facilitate the differentiation of undifferentiated cells in mature osteoblasts . In turn, osteoblasts are a terminally differentiated form of preosteoblasts (pre‐Obs) and are responsible for bone formation by sequentially producing bone matrix proteins, which subsequently induce tissue mineralization …”

Layered Double Hydroxides Are Promising Nanomaterials for Tissue Bioengineering Application

“…ECM remodeling was also required during fibroblast adaptation to both compositions of LDHs at a dynamic turnover manner, and this process might favor cell proliferation and could be governed by inflammatory and sonic hedgehog (Shh) signals . In turn, Shh is related to a multitude of cell fates and recently it has been related with inflammation process by triggering anti‐inflammatory responses .…”

“…Regrettably, serious preclinical concerns remain, the signaling cross talk with cells and biomaterials proposes are still the challenge for this promising therapeutic modality . Thus, we have explored and suggested in vitro approaches to predict the quality of the cell/biomaterial interaction, suggesting the intracellular signaling as a preliminary and reliable tool on this matter . Overall, we have shown that cells actively respond to biomaterials modifying their morphology as an immediate consequence of a well‐orchestrated cytoskeleton rearrangement upon integrin activation, where the phosphorylation of cofilin drives actin filaments rearrangement and governs cell adaptation on the substrate.…”