Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

<p>Abstract</p> <p>Background</p> <p>Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated.</p> <p>Results</p> <p>We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection.</p> <p>Conclusion</p> <p>These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.</p

Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-3

At weeks 1, 3, 5, 7, 9, 11, and 13 following HTLV-1 infection in the presence or absence of IL-2 compared to day 0; controls 1 and 4 are PBMC-1 and PBMC-2; controls 2 and 5 are PBMC-1 and PBMC-2 stimulated with PHA; controls 3 and 6 are PBMC-1 and PBMC-2 stimulated with IL-2 for one week. ANOVA with Dunnett's tests were used to analyze the data from PTH1R RT-PCR quantification (bar graph shown at the bottom of the panel). The PTH1R levels were significantly greater at weeks 5, 7, 9, and 13 (p < 0.05; indicated by asterisks in the figure). PTH1R expression in HTLV-1-infected T-cells and ATLL cells. Lanes represent: (1) Jurkat (2) MT-2 (3) SLB-1 (4) HUT102 (5) C8166 (6) MET-1 (7) RV-ATL (8) HT-1RV cells. The data showed that PTH1R expression was very low or absent in the ATLL cells (MET-1 and RV-ATL) compared to HTLV-1-infected T-cell lines (MT-2, SLB-1 and HT-1RV). Jurkat T-cells were used as a negative control. βM was used a loading control.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-4

Malized to human βM gene expression.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-6

The results showed that MIP-1α expression was up-regulated in PBMCs following stimulation with PHA or IL-2. HTLV-1 infection markedly up-regulated MIP-1α expression in the first week after infection which demonstrated that HTLV-1 infection activated the lymphocytes. MIP-1α levels were significantly different across groups and time. Overall, MIP-1α levels significantly decreased over time (p < 0.0001). After using Dunnett's method to adjust for multiple comparisons, the HTLV-1-treated groups had significantly higher MIP-1α levels than the PBMC group (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-7

(0–13 weeks after co-cultivation) and growth curves are shown. PBMCs were infected with HTLV-1 in the presence of IL-2 (10 U/mL; supplemented from day 1 following HTLV-1 infection) or in the absence of IL-2. PBMCs with no stimulation, PBMCs stimulated with PHA and IL-2 irradiated SLB-1 cells served as controls. The results showed that only HTLV-1-infected cells continued to proliferate beyond 6 weeks in culture. Viable cell numbers were significantly different over time between treatment groups (p < 0.0001). While the PBMC+HTLV-1+IL-2 group cell numbers increased slightly over time, the remaining group cell numbers decreased over time, but the PBMC+HTLV-1 group cell numbers decreased only slightly. After using Dunnett's method to adjust for multiple comparisons, the HTLV-1-treated groups both had significantly higher cell numbers than the PBMC (control) group (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-5

12, p13, p30, HBZ, Rex and Tax. The quantity of the expression plasmid is indicated in μg. Bars represent the mean ± SD of three independent samples. Relative Luc/Gal units were significantly different across groups (p = 0.0002). After adjusting for multiple times of comparison, P2/P3Luc+Rex group and P2/P3Luc+ACH group had significantly greater relative Luc/Gal units than the P2/P3Luc group (p = 0.0006, p = 0.0012, respectively; indicated by asterisks in the figure).<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation-2

Ene expression. Specific PTHrP-promoter initiated transcripts are shown for 0, 3, 7 and 13 weeks post co-culture in the presence of IL-2 (A), in the absence of IL-2 (B) and for MT-2 cells (C). The data showed that PTHrP was up-regulated in PBMCs following HTLV-1 infection by the activation of both the P2 and P3 promoters.<p><b>Copyright information:</b></p><p>Taken from "Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation"</p><p>http://www.retrovirology.com/content/5/1/46</p><p>Retrovirology 2008;5():46-46.</p><p>Published online 9 Jun 2008</p><p>PMCID:PMC2435116.</p><p></p