Calcium/calmodulin‐dependent kinase II regulates notch‐1 signaling in prostate cancer cells

Notch signaling is associated with prostate osteoblastic bone metastases and calcium/calmodulin‐dependent kinase II (CaMKII) is associated with osteoblastogenesis of human mesenchymal stem cells. Here we show that prostate cancer cell lines C4‐2B and PC3, both derived from bone metastases and express Notch‐1, have all four isoforms of CaMKII (α, β, γ, δ). In contrast, prostate cancer cell lines LNcaP and DU145, which are not derived from bone metastases and lack the Notch‐1 receptor, both lack the alpha isoform of CaMKII. In addition, DU145 cells also lack the β‐isoform. In C4‐2B cells, inhibition of CaMKII by KN93 or γ‐secretase by L‐685,458 inhibited the formation of the cleaved form of Notch‐1 thus inhibiting Notch signaling. KN93 inhibited down stream Notch‐1 signaling including Hes‐1 gene expression, Hes‐1 promoter activity, and c‐Myc expression. In addition, both KN93 and L‐685,458 inhibited proliferation and Matrigel invasion by C4‐2B cells. The activity of γ‐secretase was unaffected by KN93 but markedly inhibited by L‐685,458. Inhibition of the expression of α, β, or γ‐isoform by siRNA did not affect Hes‐1 gene expression, however when expression of one isoform was inhibited by siRNA, there were compensatory changes in the expression of the other isoforms. Over‐expression of CaMKII‐α increased Hes‐1 expression, consistent with Notch‐1 signaling being at least partially dependent upon CaMKII. This unique crosstalk between CaMKII and Notch‐1 pathways provides new insight into Notch signaling and potentially provides new targets for pharmacotherapeutics. J. Cell. Biochem. 106: 25–32, 2009. © 2008 Wiley‐Liss, Inc.     

Jmjd3 activates Mash1 gene in RA‐induced neuronal differentiation of P19 cells

Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri‐methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix‐loop‐helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA‐treated P19 cells. Over‐expression of Jmjd3 in P19 cells also significantly enhances the RA‐induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA‐induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co‐Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA‐induced neuronal differentiation of P19 cells. J. Cell. Biochem. 110: 1457–1463, 2010. © 2010 Wiley‐Liss, Inc.     

Porphyromonas gingivalis lipopolysaccharide inhibits the osteoblastic differentiation of preosteoblasts by activating Notch1 signaling

Although Porphyromonas gingivalis lipopolysaccharide (P‐LPS) is known to inhibit osteoblast differentiation, the exact molecular mechanisms underlying this phenomenon remain unclear. Here, we investigated the role of Notch signaling in the osteoblastic differentiation of both MC3T3E‐1 cells and primary mouse bone marrow stromal cells (BMSCs). P‐LPS stimulation activated the Notch1 signaling cascade and increased expression of the Notch target genes HES1 and HEY1. P‐LPS can also act as an inhibitor because it is capable of suppressing Wnt/β‐catenin signaling in preosteoblasts by decreasing both glycogen synthase kinase‐3β (GSK‐3β) phosphorylation and the expression of nuclear β‐catenin. These effects were rescued, however, by inhibiting Notch1 signaling. Furthermore, P‐LPS treatment inhibited osteoblast differentiation in preosteoblasts as demonstrated by reductions in alkaline phosphatase activity, osteoblast gene expression, and mineralization, all of which were rescued by suppression of Notch1 signaling. Moreover, inhibition of GSK‐3β, HES1, or HEY1 partially reversed the P‐LPS‐induced inhibition of osteoblast differentiation. Together, these findings suggest that P‐LPS inhibits osteoblast differentiation by promoting the expression of Notch target genes and suppressing canonical Wnt/β‐catenin signaling. J. Cell. Physiol. 225: 106–114, 2010. © 2010 Wiley‐Liss, Inc.