The correlation of TRPM1 (Melastatin) mRNA expression with microphthalmia-associated transcription factor (MITF) and other melanogenesis-related proteins in normal and pathological skin, hair follicles and melanocytic nevi.
BACKGROUND
Melastatin (TRPM1), a.k.a. transient receptor potential cation channel, subfamily M, member 1 (TRPM-1) regulates melanocyte differentiation and proliferation. TRPM1 is transcriptionally regulated by the essential melanocyte transcription factor MITF (microphthalmia-associated transcription factor). For the most part, MITF expression is preserved during melanoma progression, while TRPM1 mRNA expression decreases or is completely lost. The loss of TRPM1 is associated with melanomas that are more aggressive.


OBJECTIVE
To assess the relationship between TRPM1 mRNA expression and the expression of MITF and nine other markers of melanocytes and melanin-related proteins by immunohistochemistry in normal skin, scars, hair follicles and ordinary melanocytic nevi.


METHODS
Samples of normal skin (n = 102; from tumor excisions and plastic procedures), scars (n = 5; from re-excision specimens) and compound melanocytic nevi (n = 4) were evaluated for the presence of TRPM1 mRNA transcripts as detected by chromogenic in situ hybridization (CISH). Immunohistochemical techniques were used to detect melanin-related proteins including: MITF, S100 protein, Mart-1, tyrosinase, Mel5, HMB45, tyrosinase-related protein-1 (TRP1), TRP2 and alpha-melanocyte stimulating hormone (alphaMSH). The labeling index (LI) was defined as the number of intraepidermal cells expressing mRNA or protein per one hundred basal keratinocytes.


RESULTS
A wide range of LI was found for all markers (0-33 positive cells/100 keratinocytes). When these LI were compared, no significant differences in the expression of MITF, S100, Mart1, tyrosinase proteins and TRPM1 mRNA were identified. The LI for TRPM1 mRNA expression ranged from 74% of that for MITF to 86% for tyrosinase. The LI for TRP-1, TRP-2 and Mel5 was similar to that of TRPM1, while HMB-45 had a significantly lower LI than all other markers. TRPM1 mRNA correlated most tightly with MITF and tyrosinase expression (r = 0.81 and 0.68, respectively, both p = 0.0001). Likewise, the strongest correlation among all the melanin-related proteins existed between tyrosinase and MITF (r = 0.79, p = 0.0001). There was variable expression of melanin-related proteins when LI were analyzed by anatomic site, patient age, extent of sun-damage and proximity to a melanocytic tumor. Anogenital skin showed the highest and acral skin the lowest LI for TRPM1, MITF, S100 protein, Tyrosinase, Mel5 and HMB45. Advanced age (> 60 years) was associated with decreased TRPM1 expression. Sun-damaged skin exhibited significantly increased LI as measured by MITF, S100 protein, Mart1, tyrosinase and HMB-45, but no differences for TRPM1. However, the MITF-TRPM1 differential (i.e. MITF LI-TRPM1 LI = MITF+TRPM1--melanocytes) was significantly increased in site-matched skin (4.6 +/- 4.4 vs. 1.5 +/- 2.5, p = 0.01). There was a suggestion of reduced LI in normal skin in the proximity of melanoma (from melanoma re-excision specimens) for S100, HMB45 and TRPM1 mRNA. TRPM1 LI was significantly decreased in scars compared to normal skin (5.6 +/- 1.4 vs. 9.7 +/- 4.3, p = 0.02), this was reflected in an increase in the MITF-TRPM1 differential (9.6 +/- 7.5 vs. 3.2 +/- 3.1, p = 0.0001). MITF LI were consistently higher than MSLN LI at all levels of the hair follicle; notably, MITF was expressed by isthmic-bulge cells. In ordinary melanocytic nevi, MITF and TRPM1 expression decreased with melanocyte descent: there was more signal for both markers in superficial epithelioid type A melanocytes than deeper type C melanocytes.


CONCLUSIONS
By CISH, TRPM1 mRNA expression is specific for melanocytes and strongly associated with MITF and tyrosinase expression, the latter implicating a mature melanocyte phenotype. However, in normal skin, TRPM1 mRNA expression appears to be dynamic, labeling most but not all melanocytes, with variable expression ostensibly related to local environmental factors.
Lu
Song
S
Division of Dermatology, Albany Medical College MC-81, 47 New Scotland Avenue, Albany, NY 12208, USA.
Slominski
Andrzej
A
Yang
Sung-Eun
SE
Sheehan
Christine
C
Ross
Jeffrey
J
Carlson
J Andrew
JA
D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002921:Cicatrix / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D004817:Epidermis / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018859:Hair Follicle / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D008297:Male; D008544:Melanocytes / Q000166:cytology / Q000502:physiology; D000090204:Mesothelin; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009508:Nevus, Pigmented / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D012333:RNA, Messenger / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D050053:TRPM Cation Channels / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult; D000521:alpha-MSH / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; C497234:MITF protein, human; C000715527:MSLN protein, human; D051739:Microphthalmia-Associated Transcription Factor; D012333:RNA, Messenger; D050053:TRPM Cation Channels; C492059:TRPM1 protein, human; D000521:alpha-MSH; D000090204:Mesothelin