We analyzed the effect of migration through inhibition of HPSE activity by using transwell assay, and the result leave a message showed that the addition of SDF 1 significantly increased the migratory cell count. While OGT2115 fur ther increased the Inhibitors,Modulators,Libraries migratory cells significantly, CXCR4 inhibitor significantly decreased the migratory cells re gardless of whether HPSE was inhibited. Inhibitors,Modulators,Libraries These results indicated that the inhibition of HPSE activity enhanced chemotaxis and the blocking of CXCR4 significantly decreased this chemotaxis indicating that HPSE modulates BM MSCs homing via SDF 1/CXCR4 signaling axis. To further demonstrate the specificity of the effect of OGT2115 on migration, the transwell migration assay with SDF 1 was repeated with or without the presence of OGT2115 and/or mouse recombinant HPSE1.
In accordance with our hypothesis, the addition of mouse recombinant HPSE1 demonstrated a trend of reduced migratory BM MSCs similarly to the CXCR4 inhibitor and significantly reversed the potentiation of migration by OGT2115 indicating that the effect Inhibitors,Modulators,Libraries of OGT2115 is specifically through the inhibition of HPSE1 and that HPSE negatively modulates the migration of BM MSCs. Like proliferation capacity, the migration ability of BM MSCs also decreased along the serial passages. We therefore also performed transwell migration assay with SDF 1 on P2 and P6 BM MSCs. Consist ent with the experiments done with P4 BM MSCs, inhibition on endogenous HPSE po tentiated the cell migration at both P2 and P6 BM MSCs indication that the effect of HPSE on modulating BM MSCs migration persist through serial passages albeit the migration capacity decreased in later passages.
Previous studies indicated that HS GAGs interact with SDF 1 directly and cell surface HSPGs mediate the SDF 1/CXCR4 Inhibitors,Modulators,Libraries binding and signaling. We would like to know whether gene transactivation is also involved. To answer this question, we analyzed migration related genes including Sdf1, Cxcr7 and Cxcr4, and found that the expression Inhibitors,Modulators,Libraries level of Cxcr4 increased significantly under the treatment of HPSE inhibitor sug gesting that HPSE also modulates BM MSCs via a gene transactivation mechanism. Heparanase participated in chromatin remodeling Previous studies indicated that nuclear HPSE and hepa ran sulfate glycosaminoglycans might participate in the transcriptional regulation via the modulation of the enzymatic activities of histone acetyltrasnferases such as p300 and DNA topoisomerase I.
Our re sults demonstrated the altered gene expression patterns under the inhibition of HPSE. We hence hypothesized that HPSE could participate in the maintenance of self renewal of BM MSCs, at least par tially, via this thoroughly intranuclear mechanism involving in chro matin remodeling. To this end, universal histone H3 and histone H4 acetylation in BM MSCs were quantified by western blot.