Human melastatin 1 (TRPM1) is regulated by MITF and produces multiple polypeptide isoforms in melanocytes and melanoma

Abstract

Melastatin 1 (MLSN1), originally identified as melanoma metastasis suppressor, represents a TRPM subfamily of transient receptor potential (TRP) proteins which serve diverse biological roles in a wide variety of cell types. Down-regulation of MLSN1 expression in human cutaneous melanoma, as indicated by in situ hybridization, appears to be a prognostic marker for melanoma metastasis. However, the exact physiological function(s) of MLSN1, the mechanism(s) involved in the regulation of its expression and its role in melanoma tumour progression are not yet clear. In this study, we identified a 654 bp upstream sequence of MLSN1, containing four E boxes (E1-E4), including an 11 bp M box (E4), that is sufficient for melanocyte-specific transcription and activation by the melanocyte transcription factor MITF (a bHLH-zip factor). Deletion analysis showed that the two distal E boxes (E3 and E4) in the MLSN1 promoter are required for both its activation by MITF and its constitutive activity in melanoma cells. Western blot analysis using polyclonal rabbit anti-human MLSN1 antibodies identified several polypeptides, presumably generated by both alternative splicing of MLSN1 messenger RNA (mRNA) and proteolytic cleavage, in both melanocytes and metastatic melanoma cells. Thus, multiple mechanisms appear to regulate MLSN1 expression in melanocytes and melanoma cells.

Transcriptional regulation and processing increase the functional variability of TRPM channels

“…This promoter is capable to drive reporter gene expression in mouse and human melanoma cells. Recently, it could be shown that MITF is an essential transcription factor for melanocytic development and a major transcriptional regulator of TRPM1 expression through its interaction within the proximal promoter region Zhiqi et al 2004). RT-PCR analysis of mouse (Duncan et al 1998) and Northern blots of rat tissues (Fig.…”

“…2a) contains transcriptional regulatory elements unique to genes (Sakura and Ashcroft 1997;Chang et al 1997;Wang et al 1999;den Dekker et al 2001;Yang et al 2002;Antoniotti et al 2002) TRPC 2 (Berg et al 1997;Wissenbach et al 1998;Vannier et al 1999;Hofmann et al 2000;Chu et al 2002Chu et al , 2004Yildirim et al 2003) TRPC 3 (Ohki et al 2000;Yang et al 2002) TRPC 4 (Freichel et al 1998;Walker et al 2001Walker et al , 2002Mery et al 2001;Schaefer et al 2002;Yang et al 2002;Qian et al 2002;Satoh et al 2002) TRPC 6 (Philipp et al 2000Zhang and Saffen 2001) TRPC 7 (Walker et al 2001) TRPV1* (Schumacher et al 2000;Sanchez et al 2001;Xue et al 2001;Wang et al 2004) TRPM1 (Fang and Setaluri 2000;Xu et al 2001;Zhiqi et al 2004); this manuscript TRPM2 (Wehage et al 2002;Zhang et al 2003a) TRPM3 (Lee et al 2003) TRPM4 (Launay et al 2002;Nilius et al 2003;Murakami et al 2003) TRPM5 (Prawitt et al 2000) TRPM6 …”

Malignant melanoma: genetics and therapeutics in the genomic era

“…Pigmentation-associated transcriptional targets of MITF include the pigment enzyme genes tyrosinase, TRP1, TRP2/Dct, which were recognized to contain an evolutionarily conserved consensus promoter/enhancer element that matches the MITF recognition sequence (Bentley et al 1994;Hemesath et al 1994;Yasumoto et al 1994). Additional differentiation-associated genes include Pmel17/silver/gp100 (which encodes the melanoma diagnostic epitope HMB45) (Halaban et al 1996;Baxter and Pavan 2003;Du et al 2003), MelanA/Mart1 , Melastatin (TRPM1) (Miller et al 2004;Zhiqi et al 2004), AIM1 (ocular albinism 4 gene) (Du and Fisher 2002), Ocular albinism 1 gene (OA1) (Vetrini et al 2004), VMD2 (Esumi et al 2004), HIF1␣ (Busca et al 2005), Plasminogen activator inhibitor-1 (Murakami et al 2006), numerous mast cell targets of MITF including Prostaglandin D2, multiple mast cell genes including proteases, various adhesion molecules and others (see Ito et al 2004;Morii et al 2004;Takeda et al 2006 and references therein), and melanocortin 1 receptor (MC1R) in mast cells and possibly in melanocytes (Adachi et al 2000;Smith et al 2001;Aoki and Moro 2002). Numerous additional genes are being identified through expression microarrays, and are liable to reflect the multiple steps involved between signaling, expressing, packaging, and exporting pigment.…”

TRPM1 (transient receptor potential cation channel, subfamily M, member 1)

“…The TRPM1 gene encodes a 5.4kb (5428bp) mRNA transcript (Hunter et al, 1998;Fang and Setaluri, 2000). The promoter region of this gene contains 4 consensus binding sites for the microphthalmia-associated transcription factor (MITF), one of those binding site includes an M box, a motif shared by pigmentation genes (Hunter et al, 1998;Miller et al, 2004;Zhiqi et al, 2004). A 1-kb PvuII fragment from this region is capable of driving reporter gene expression in mouse and human melanoma cells.…”

“…The shorter N-terminal isoform, TRPM1 (MLSN1-S), lacking the transmembrane domains, encodes for 500 aa protein (Fang and Setaluri, 2000). Rabbit polyclonal antibodies generated against the N-terminal part of TRPM1 detected proteins with molecular weights of 120 kDa and several minor bands ranging from 35 to 240kDa, including a doublet at 45kDa in primary neonatal melanocytes (Zhiqi et al, 2004).…”