Date of Award

1996

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Beverly Delidow

Second Advisor

Susan Jackman

Third Advisor

Vernon Reichenbecher

Fourth Advisor

Monica Valentovic

Fifth Advisor

Richard M. Niles

Sixth Advisor

Leonard J. Deutsch

Abstract

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.

Subject(s)

Tretinoin.

Retinoids.

Protein kinase C.

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