Background: Sonic hedgehog (Shh) and components of its signalling pathway have been identified in human prostate carcinoma and increased levels of their expression appear to correlate with disease progression and metastasis. The mechanism through which Shh signalling could promote metastasis in bone, the most common site for prostate carcinoma metastasis, has not yet been investigated. The present study determined the effect of Shh signalling between prostate cancer cells and pre-osteoblasts on osteoblast differentiation, a requisite process for new bone formation that characterizes prostate carcinoma metastasis.
BackgroundInduction of osteoblast differentiation by paracrine Sonic hedgehog (Shh) signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts.ResultsIn the presence of exogenous ascorbic acid (AA), a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells) formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis inhibitor 3,4-dehydro-L-proline.ConclusionsMatrix collagen, formed by osteoblasts in the presence of AA, potentiates Shh signaling between Shh-expressing prostate cancer cells and osteoblasts. Collagen and Shh signaling exert a synergistic effect on osteoblast differentiation, a defining event in prostate carcinoma bone metastasis. Investigations into paracrine interactions among prostate cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins advance our understanding of mechanisms contributing to prostate cancer bone metastasis.
IL-12 is a 70 kD heterodimeric cytokine that consists of two subunits, p40 and p35. The p40 subunit can also form p80 homodimers, or it can associate with p19 to form IL-23. IL-12 is known to be induced in dendritic cells (DCs) by pathogens, through toll-like receptors (TLRs), and by activated T cells, through CD40-CD40L engagement. To investigate the response of antigen presenting cells (APCs) to naïve T cells, we cocultured DCs and OVA peptide with CD4+ CD62L+ OVA-specific T cells. Naïve T cells induced immature DCs to secrete p40, but not IL-12, during antigen presentation, in sharp contrast to the nearly exclusive secretion of IL-12 heterodimers by DCs presenting to CD62L− T cells. Maximal secretion of p40 occurred within 24 hr after T cells were added to the APCs. During this time period, CD40 and CD40L expression increased on DCs and T cells, respectively, but no significant difference was seen in the expression of CD40L on CD62L+ and CD62L− T cells. IL-23 induction was measured by real-time RT-PCR and by ELISA. IL-23 was not induced by naïve T cells unless the DCs were activated. Western blot analysis will reveal whether the excess p40 induced by naïve T cells is secreted primarily as monomers or homodimers. Future experiments will also address the functional significance of excess p40 secretion during the encounter of immature APCs with naïve T cells. (Supported by NIH grant S06-GM08043 to K.S.)
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