Mechanisms Regulating CXCR4 Expression in Myeloma Cells.

The chemokine receptor CXCR4 plays roles in the homing of myeloma cells to the bone marrow (BM) and in the progression of the disease. However, the regulation of CXCR4 expression in myeloma cells is poorly defined. This study investigated the mechanisms regulating CXCR4 expression in myeloma cells. RPMI8226 and U266 myeloma cells strongly expressed CXCR4 on the cell surface, whereas ARH77 myeloma cells expressed minimal CXCR4 on the cell surface, as determined by flow cytometry using three different monoclonal antibodies to CXCR4. However, Western blot analysis, flow cytometry after permeabilization, and immunofluorescence staining reveled that ARH77 cells have abundant CXCR4 in the cytoplasm, in amounts similar to those in RPMI8226 and U266 cells. The cell surface expression of CXCR4 in primary CD138+ cells obtained from the BM of multiple myeloma patients differed among different specimens. Similar to myeloma cell lines, the primary myeloma cells that expressed minimal CXCR4 on the cell surface had abundant CXCR4 in the cytoplasm. The transmigration of the cells induced by stromal cell-derived factor-1 (SDF-1) was correlated with the cell surface expression of CXCR4, indicating that only the CXCR4 on the cell surface is functional. These findings suggest that myeloma cells have their own intrinsic mechanisms for regulating CXCR4 expression on the cell surface. In all three myeloma cell lines and in some primary BM CD138+ cells, dexamethasone (Dex) enhanced CXCR4 expression both in the cytoplasm and on the cell surface while downregulating SDF-1; this led to enhanced cell migration in response to SDF-1. Cell surface CXCR4 expression was more prominent in annexin V-positive apoptotic cells. VEGF and proinflammatory cytokines, including TNF-α and TGF-β¹, also upregulated the cell surface expression of CXCR4 in RPMI8226 and some primary BM CD138+ cells, but not in U266 and ARH77 cells. Myeloma cells, including all the three cell lines and some primary BM CD138+ cells, incubated under hypoxic conditions (1% O₂) exhibited upregulation of CXCR4 both in the cytoplasm and on the cell surface. Again, surface CXCR4 expression was stronger in apoptotic cells than in non-apoptotic cells, suggesting that the upregulation of CXCR4 is a counter-regulatory phenomenon for stimuli causing cell damage. As proven previously for other cell types, hypoxia induced the accumulation of HIF-1α in myeloma cells. Topotecan, which inhibits HIF-1α, attenuated the hypoxia-induced upregulation of CXCR4, whereas the proteasome inhibitor bortezomib slightly enhanced it. Dex, VEGF, TNF-α, and TGF-β¹ all induced the accumulation of HIF-1α in RPMI8226 and some primary BM CD138+ cells. In addition, the effects of topotecan and bortezomib under hypoxic conditions were observed in the change of CXCR4 expression mediated by Dex and cytokines. These results indicate that complex intrinsic and extrinsic mechanisms regulate CXCR4 expression in myeloma cells and suggest that HIF-1α is a common regulatory molecule, at least in the extrinsic mechanisms.