Oncostatin M regulates osteogenic differentiation of murine adipose-derived mesenchymal progenitor cells through a PKCdelta-dependent mechanism.

Oncostatin M (OSM) is an IL-6/LIF family cytokine that influences mesenchymal progenitor differentiation; however, the mechanisms of this activity have not been fully elucidated. Using uncommitted murine adipose tissue-derived mesenchymal progenitors, we have examined mechanisms of OSM-induced osteogenesis. Murine OSM (mOSM) induced osteogenic differentiation to a greater degree than interleukin (IL)-6 and other members of the gp130 cytokine family, promoting extracellular matrix mineralization as indicated by Alizarin Red S staining. mOSM also increased expression of osteogenesis-associated gene products BMP4, BMP7, Runx-2, and osteocalcin as assessed by immunoblotting and real-time quantitative PCR. Additionally, protein kinase C (PKC) delta activity was upregulated in response to OSM stimulation, and to a greater degree than IL-6. Knockdown of PKCdelta expression by use of RNA interference (RNAi) reduced OSM-mediated osteogenic differentiation and decreased expression of Runx-2. These findings suggest that OSM differentially promotes osteogenesis in non-committed mesenchymal progenitors relative to other gp130 cytokines. This activity correlates with selective activation of PKCdelta in OSM-treated cells, indicating that OSM-induced osteogenesis and upregulation of osteogenic gene products require activity of PKCdelta.

Oncostatin M induction of eotaxin-1 expression requires the convergence of PI3'K and ERK1/2 MAPK signal transduction pathways.

Oncostatin M (OSM) is an IL-6/LIF cytokine family member whose role has been identified in a range of biological activities in vitro, including upregulation of inflammatory gene expression and regulation of connective tissue metabolism. Previously, we identified murine OSM (mOSM) as an inducer of the eosinophil-chemoattractant protein eotaxin-1, both in vitro using fibroblast cell lines and in vivo from mouse lung tissues. Using the NIH 3T3 cell line, we demonstrate the requirement of PI3'K activation for mOSM induction of eotaxin-1 through inhibition of both mOSM-stimulated mRNA and protein expression using the PI3'K antagonist LY294002. By assessment of phosphorylation of the downstream mediator Akt, we show mOSM to be differentially capable of activating PI3'K relative to the related gp130-utilizing cytokine IL-6. Assessment of eotaxin-1 gene expression utilizing PKB/Akt mutant-transfected NIH 3T3 cell lines demonstrated Akt is not involved in upstream regulation of eotaxin-1 through mOSM, indicating an alternate kinase pathway downstream of PI3'K may be involved. We demonstrate that mOSM stimulation of expression of eotaxin-1 is reduced by PD98059, a MAPK kinase inhibitor selective for MEK1. Both LY294002 and PD98059 attenuated mOSM-induced phosphorylation of ERK1/2 MAP kinase and also reduced binding of an AP-1 responsive promoter element, a transcriptional complex known to be MAPK-sensitive. Further, LY294002 pretreatment reduced mOSM-stimulated expression of the downstream AP-1 co-factor JunB, while PD98059 reduced levels of JunB as well as c-Fos. These results provide evidence for a previously unidentified signaling mechanism utilized by mOSM for the induction of eotaxin-1.

Oncostatin M-induced IL-6 expression in murine fibroblasts requires the activation of protein kinase Cdelta.

Oncostatin M (OSM) is an IL-6/LIF cytokine family member whose role has been identified in a range of biological activities in vitro, including up-regulation of inflammatory gene expression and regulation of connective tissue metabolism. However, the mechanisms through which OSM regulates cellular responses are not completely understood. In this study, we show that activation of the calcium-independent or novel protein kinase C (PKC) isoform PKCdelta is a critical event during OSM-mediated up-regulation of IL-6 expression in murine fibroblasts. The pan-PKC inhibitor GF109203X (bisindolylmaleimide I) reduced secretion of IL-6; however, use of Go6976, an inhibitor of calcium-dependent PKC enzymes, did not. The PKCdelta-selective inhibitory compound rottlerin abrogated expression of IL-6 transcript and protein, but only reduced PKCdelta activity when used at higher concentrations as determined by kinase activity assay, suggesting rottlerin may inhibit IL-6 expression in a PKCdelta-independent manner. However, silencing of PKCdelta protein expression, but not the related novel isoform PKCepsilon, by use of RNA interference (i.e., small interfering RNA) demonstrated that PKCdelta is required for murine OSM (mOSM) induction of IL-6 protein secretion. Furthermore, inhibition of PI3K by use of LY294002 reduces expression of IL-6 at both the mRNA and protein level in murine fibroblasts, and we suggest that PI3K is required for activation of PKCdelta. Knockdown of phosphoinositide-dependent kinases PDK-1 or Akt1 using small interfering RNA strategies did not influence mOSM-induced IL-6 expression, suggesting mOSM uses a PI3K-PKCdelta pathway of activation independent of these kinases. Our findings illustrate a novel signaling network used by mOSM that may be important for its mediation of inflammatory processes.