Interplay between MITF, PIAS3, and STAT3 in Mast Cells and Melanocytes

Abstract

Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function in mast cells and melanocytes. In the present study, we explored the MITF-PIAS3-STAT3 network of interactions, how these interactions regulate gene expression, and how cytokine-mediated phosphorylation of MITF and STAT3 is involved in the in vivo interplay between these three proteins. In NIH 3T3 cells stimulated via gp130 receptor, transfected MITF was found to be phosphorylated at S409. Such phosphorylation of MITF leads to PIAS3 dissociation from MITF and its association with STAT3. Activation of mouse melanoma and mast cells through gp130 or c-Kit receptors induced the mobilization of PIAS3 from MITF to STAT3. In mast cells derived from MITF di/di mice, whose MITF lacks the Zip domain (PIAS3-binding domain), we found downregulation in mRNA levels of genes regulated by either MITF or STAT3. This regulatory mechanism is of considerable importance since it is likely to advance the deciphering of a role for MITF and STAT3 in mast cells and melanocytes.Microphthalmia transcription factor (MITF) is a basic helixloop-helix leucine zipper (bHLH-Zip) DNA-binding protein (17). Its gene resides at the mi locus in mice (19), and mutation of this gene results in deafness, bone loss, small eyes, and poorly pigmented eyes and skin (32). The primary cell types affected in MITF-deficient mice are mast cells, osteoclasts, and melanocytes (32). In humans, mutation in this gene causes Waardenburg syndrome type II (40). MITF regulates the expression of mouse mast cell protease 6 (mMCP) (35), mMCP5 (33), c-Kit (20), p75 nerve growth factor (33), granzyme B (9), and tryptophan hydroxylase (21). MITF regulates gene transcription by binding to E-box-type enhancers in the 5Ј-flanking regions of MITF-responsive genes (35). Like many other DNA-binding proteins, the transcription-enhancing activity of MITF is influenced in a complex manner by an array of different intracellular proteins. We previously identified two MITF-interacting proteins, PKCI/Hint (37) and PIAS3 (27), by using the yeast two-hybrid system with the bHLH-Zip domain as a bait. These two MITF-associated proteins were shown to be repressors of MITF transcriptional activity (27,37). We have also shown by pull-down assay and by using cells derived from MITF truncated mice (di/di) that the MITF Zip domain plays an essential role in the interaction between MITF and PIAS3 (28).Three serine sites for MITF phosphorylation have been reported (14,42,46). Phosphorylation of S73 (14) and S409 (46) occurs as a result of kit ligand stimulation and activation of mitogen-activated protein kinase and Rsk-1, respectively. These serine sites are of interest since, upon their phosphorylation, MITF transcriptional activity is upregulated, and this phosphorylation also serves as a signal for the degradation of MITF by ubiquitin-dependent proteolysis in melanocytes (46).In our previous study, we observed that phosphorylation of MITF at S73 and S409...

A Specific Epitope of Protein Inhibitor of Activated STAT3 Is Responsible for the Induction of Apoptosis in Rat Transformed Mast Cells

“…For instance, the ring finger domain has been shown to be important for PIAS3's activity as a sumo-E3 ligase (21,23). We have found that PIAS3 functions in vivo as a key molecule in suppressing the transcriptional activity of both MITF and STAT3 (10,24,25). More specifically, we demonstrated that following cellular activation, PIAS3 is released from MITF and binds to STAT3 (25).…”

Protein Inhibitor of Activated STAT 3 Modulates Osteoclastogenesis by Down-Regulation of NFATc1 and Osteoclast-Associated Receptor

“…In the nucleus of resting cells, unphosphorylated Mitf is inactivated by association with PIAS3. Activation of the gp130 receptor or c-Kit receptor in melanoma and mast cells induces Mitf phosphorylation and results in the release of PIAS3, which then binds STAT3 (37,38). This indicates that the phosphorylation and dephosphorylation of Mitf is a key step in the regulation of interaction between Mitf, PIAS3, and STAT3.…”

The Intracellular Delivery of a Recombinant Peptide Derived from the Acidic Domain of PIAS3 Inhibits STAT3 Transactivation and Induces Tumor Cell Death

“…This does not confirm previously published studies of the group of Razin. These investigators described the NH 2 terminus of mPIAS3 as the major STAT3 interaction domain (29,38). The discrepancies between the data might be due to different methods used in the interaction analyses, the use of different cell types, and different PIAS3 variants.…”

“…It was therefore reasonable to postulate that PIAS3 and STAT3 might interact through similar domains. However, a previous report showed that an NH 2 -terminal fragment of mPIAS3, comprising amino acid positions 82 to 132, interacts with STAT3 in murine cell lines (15,29). We performed yeast two-hybrid analyses with the NH 2 terminal (amino acid positions 1-319) and the COOH terminal (amino acid positions 320-619) fragments of hPIAS3.…”