Date of Award
Joan C. Edwards School of Medicine
Type of Degree
Richard M. Niles
Leonard J. Deutsch
Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells. This differentiation is accompanied by an increase in protein kinase Ca (PKCα) protein level and selective enrichment in nuclear-associated PKCα. PKC is thought to regulate gene expression through the TPA response element (TRE). This element is specifically recognized by the AP-1 transcription factor composed of jun and fos family members. In this study, I have analyzed the effect of RA on the expression and function of AP-1 in B16 mouse melanoma cells. Transient transfection analysis of B16 cells using leuciferase reporter gene constructs with or without AP-1 elements indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity in a concentration-dependent manner. RA did not change the expression (mRNA and protein) of jun family members while the expression (mRNA and protein) of c-fos was decreased. In contrast, acute phorbol dibutyrate (PDB) treatment increased c-jun and c-fos expression. Analysis of the mobility shift assay by using an oligonucleotide containing the AP-1 element suggested that two of the complexes were negatively regulated by RA. There was no significant change in the binding of the other complexes by RA. Acute PDB treatment increased the binding where as chronic treatment decreased the binding of this complex. Use of specific antibodies indicated that complexes which were decreased by RA and increased by PDB contained fos protein. Down regulation of PKCα by chronic PDB treatment inhibited both the acute PDB and the RA-induced increase in AP-1 activity. However, the potent and selective PKC inhibitor bisindolylmaleimide inhibited the PDB induced increase in AP-1 activity but had no effect on the RA-induced increase in AP-1 activity. Our results suggest that the role played by PKC in RA induced AP-1 activity is independent of its kinase activity. I also determined the role of nuclear retinoid receptors in RA-induced PKCα expression and AP-1 transcriptional activity by using receptor-specific analogs. Results suggest that RARα and RXR play an important role in RA-mediated effect on PKCα expression and AP-1 activity.
Protein kinase C.
Desai, Sejal H., "Effect of retinoic acid on the expression and function of AP-1 transcription factor in B16 mouse melanoma cells: role of protein kinase" (1996). Theses, Dissertations and Capstones. 1472.