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

Susan Jackman

Second Advisor

Michael Moore

Third Advisor

Donald Primerano

Fourth Advisor

Vernon Reichenbecher

Fifth Advisor

Richard Niles

Sixth Advisor

Leonard J. Deutsch

Abstract

The pigment cell-specific expression of tyrosinase and TRP1 has been shown to be important for the production of melanin in pigmented cells. Using a pigmented cell line, B16 mouse melanoma, we obtained evidence that PKC plays a major role in regulating melanogenesis. Chronic treatment with phorbol dibutyrate (PDBu) leads to downregulation of PKC activity and protein levels. This is accompanied by a loss of pigmentation which is correlated with a 50% reduction and a complete loss of TRP1 and tyrosinase respectively. Similar results were obtained with Northern and Western blotting indicating that PKC may regulate the steady state levels of these melanogenic proteins. Deletion analysis of the mouse tyrosinase promoter region showed that a 45 bp region ~100 bp from the transcriptional start site is essential for efficient transcription of the gene. The central part of this region co-localizes with an 11-bp motif termed the M-box. This part of the promoter i.e. the M-box is conserved in the promoter regions of melanocyte-specific genes. Using Retinoic acid (RA) and PDBu as opposing agents in regulating PKC levels and melanin biosynthesis, we demonstrate a major role for PKC in transcription of the tyrosinase gene as determined by reporter gene assays. RA increased and PDBu decreased the transcriptional activity of the pigment cell-specific tyrosinase promoter. We also found that the assembly of nuclear protein complexes at the M-box region varies with cell density. One of the complexes was found to be negatively regulated by chronic PDBu treatment. Antibody-supershift experiments showed that this PKC-regulated complex may contain microphthalmia-associated transcription factor (MITF) and retinoic acid receptors (RARs). We conclude that tyrosinase gene expression is regulated by a complex interplay of signaling mechanisms, regulated either directly or indirectly by PKC.

Subject(s)

Melanins – Synthesis.

Protein kinase C.

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