Expression dynamics of integrin α2, α3, and αV upon osteogenic differentiation of human mesenchymal stem cells.

BACKGROUND: The differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis. METHODS: To study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-ß1 (TGF-ß1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-ß1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR. RESULTS: The binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin α2, α3, and αV, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin α2 was drastically downregulated, but the expression of integrin α3 and αV was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin α3 and αV was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin αV-high hMSCs have a greater osteogenic potential than integrin αV-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin αV is more dramatically induced even in integrin αV-low hMSCs. CONCLUSION: These findings suggest that integrin α3 and αV induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.

Enhanced expression of cell-surface B-cell receptor-associated protein 31 contributes to poor survival of non-small cell lung carcinoma cells.

B-cell receptor-associated protein 31 (BAP31) is an endoplasmic reticulum (ER) membrane protein which plays a role as a molecular chaperone for the newly synthesized transmembrane proteins. BAP31 is also an important apoptosis regulator for extrinsic apoptosis induction in the ER membrane. Recent studies have shown that BAP31 is also expressed on the surface of embryonic stem cells. However, the function of cell surface BAP31 (csBAP31) still remains unclarified. In an attempt to search for surface markers on tumorspheres, here, we generated monoclonal antibodies (MAbs) against the sphere cells from the non-small cell lung carcinoma cell (NSCLC) line A549. SP1-B7, one of the MAbs, recognized csBAP31 whose expression was further increased on A549 sphere cells, as compared with A549 adherent cells. To investigate the role of csBAP31 in A549 cells, A549 adherent and sphere cells were stained with annexin V, propidium iodide, and SP1-B7. Interestingly, annexin V-high cells showed increased expression of csBAP31 as compared with annexin V-low cells. Caspase-3/7 activity was also increased in csBAP31-high cells as compared with csBAP31-low cells, suggesting that csBAP31-high cells are more sensitive to apoptosis. To further demonstrate the survival of csBAP31-positive A549 cells, csBAP31-positive and -negative A549 cells were sorted and subjected to the clonogenic survival assay. The colony number of csBAP31-positive A549 cells was decreased by approximately 1.7-fold, as compared that of csBAP31-negative A549 cells, suggesting that csBAP31-positve cells are sensitive to cell death indeed. The results suggest that enhanced expression of csBAP31 contributes to poor survival of NSCLC cells.

Cell-surface major vault protein promotes cancer progression through harboring mesenchymal and intermediate circulating tumor cells in hepatocellular carcinomas.

Circulating tumor cells (CTCs) play a major role in the metastasis and recurrence of hepatocellular carcinoma (HCC). Here, we found that major vault protein (MVP) is expressed on the surface of HCC cells and further induced under stressful environments. MVP knockdown reduces cell proliferation and induces apoptosis in HCC cells. Treatment of HCC cells with anti-MVP antibody (α-MVP) recognizing cell-surface MVP (csMVP) inhibits cell proliferation, migration, and invasion. csMVP-positive HCC cells have a higher clonogenic survival than csMVP-negative HCC cells, and treatment of HCC cells with α-MVP inhibits clonogenic survival, suggesting that csMVP contributes to HCC cell survival, migration, and invasion. The function of csMVP is mediated through mTOR, FAK, ERK and Akt signaling pathways. csMVP-positive CTCs are detected in HCC patients (89.7%) but not in healthy donors, and the number of csMVP-positive CTCs is further increased in patients with metastatic cancers. csMVP is exclusively detectable in CTCs with mesenchymal phenotype or intermediate phenotype with neither epithelial nor mesenchymal markers, suggesting that csMVP-associated survival and metastatic potential harbor CTCs with nonepithelial phenotypes. The results suggest that csMVP promotes cancer progression and serves as a surface marker for mesenchymal and intermediate CTCs in patients with HCC and metastatic cancers.