Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells.
In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent.
Yang
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Department of Surgery, University of Texas Health Science Center, San Antonio, San Antonio, Texas 78229, USA.
Yang
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J
Venkateswarlu
S
S
Ko
T
T
Brattain
M G
MG
D030201:Activin Receptors, Type I; D019138:Antibodies, Blocking / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects*; D001940:Breast / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D002117:Calcitriol / Q000031:analogs & derivatives / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002762:Cholecalciferol / Q000031:analogs & derivatives / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018167:Receptors, Calcitriol / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein; D013936:Thymidine / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology
D019138:Antibodies, Blocking; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D002762:Cholecalciferol; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D002117:Calcitriol; C078903:seocalcitol; D013936:Thymidine