Role of gap junctions between keratinocyte and melanocyte in melanogenesis

doi:10.1007/s11515-015-1381-z

Front. Biol.

2015, Vol. 10

Issue (6) : 495-502 https://doi.org/10.1007/s11515-015-1381-z

RESEARCH ARTICLE

Role of gap junctions between keratinocyte and melanocyte in melanogenesis

Divya Padma^1,²,Kapaettu Satyamoorthy¹,Kumar M.R. Bhat^2,^*(

)

1. Division of Biotechnology, School of Life Sciences, Manipal University, Manipal-576104, India
2. Department of Anatomy, Kasturba Medical College, Manipal University, Manipal-576104, India

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Abstract

The process of melanogenesis in melanocytes and the transport of melanin in the form of melanosomes to the neighboring keratinocytes are the key steps in human skin pigmentation. Keratinocytes and melanocytes interact in intricate manner to maintain the homeostasis. The present study was designed to understand the role of cell-cell interaction through the gap junctions between melanocytes and keratinocytes on melanogenesis. We show that, inhibition of the gap junctional activity between human keratinocytes and melanocytes in a coculture system using gap junction blocker lowers the expression of key regulatory genes of melanogenesis such as tyrosinase and microphthalmia-associated transcription factor (MITF). This was followed by concurrent decrease in tyrosinase protein levels and activity. Our results show the preliminary evidence for the regulation of melanogenesis in melanocytes through direct gap junctional communication by keratinocytes. Deciphering the mechanism and factors involved in the process would uncover the significance of gap junctions in melanogenesis.

Keywords melanocyte keratinocyte melanogenesis gap junctions cell communication

Corresponding Author(s): Kumar M.R. Bhat

Just Accepted Date: 30 December 2015 Online First Date: 14 January 2016 Issue Date: 26 January 2016

Cite this article:

Divya Padma,Kapaettu Satyamoorthy,Kumar M.R. Bhat. Role of gap junctions between keratinocyte and melanocyte in melanogenesis[J]. Front. Biol., 2015, 10(6): 495-502.

URL:

https://academic.hep.com.cn/fib/EN/10.1007/s11515-015-1381-z
https://academic.hep.com.cn/fib/EN/Y2015/V10/I6/495

Fig.1 Cytotoxicity of oleamide. Oleamide toxicity in (A) keratinocyte and (B) melanocytes. A concentration of oleamide above 125 µM was found to be toxic. **, p<0.01 ; ***, p<0.001 in comparison with untreated group.

Fig.2 Micrographs of stained keratinocytes and melanocytes. Human keratinocytes stained with Calcein (A) Fluorescence, (B)bright-field image. Human melanocytes stained with Dil (C) Fluorescence (D) bright-field image. Coculture of keratinocytes and melanocytes (E) stained with Calcein (fluorescence) (F) stained with Dil (fluorescence) and (G) bright-field image. White circles are marking Dil stained melanocyte interacting with Calcein stained keratinocytes, observed under (E) blue excitation filter (450-480nm) (F) green excitation filter (510–550nm) and (G) bright-field in fluorescence microscope.

Fig.3 Gap junctional activity between keratinocyte and melanocyte–flow cytometry assay. Quadrant2 (Q2) cells are double positive (positive for both Calcein and Dil) indicating the dye transfer from donor (keratinocytes) to recipient (melanocytes) cells. (A) Control, (B) + 25µM of oleamide, (C) + 50µM of oleamide, (D) + 100µM of oleamide. (E) Dose dependent decrease in the double stained cells i.e. decreased gap junctional activity was observed in oleamide treated cocultures (F). *, p<0.05 and **, p<0.01

Fig.4 Gap junctional activity between melanocytes—flow cytometric analysis. Quadrant 2 (Q2) represents the cells which aredouble positive (positive for both Calcein and Dil) which in turn indicates the dye transfer from donor melanocytes (Calcein stained cells) to recipient melanocytes (Dil stained cells). (A) Control, (B) + 25µM of oleamide, (C) + 50µM of oleamide, (D) + 100µM of oleamide. (E) Addition of oleamide to the culture containing Calcein stained andDil stained melanocytes did not show any significant changes in the gap junctional activity.

Fig.5 Effect of oleamide on transcriptional level of MITF (A) and TYR (B) in melanocytes and coculture of keratinocytes and melanocytes. No significant change was observed in theexpression level of MITF and TYR in melanocytes when treated with oleamide but in coculture of keratinocytes and melanocytes, there was a significant decrease in expression level of MITF (***, p<0.001) and tyrosinase (**, p <0.01) in comparison with untreated control group.

Fig.6 Western blot analysis to show the tyrosinase protein level. Level of tyrosinase protein in the cell lysate (A) from melanocytes alone (B) from coculture of melanocyte and keratinocytes with or without addition of oleamide. (C) Graphical representation to show the reduction in the expression of tyrosinase protein. Addition of oleamide significantly reduced the expression of tyrosinase protein in coculture of keratinocytes and melanocytes and not in the culture containing only melanocytes. The data represents the mean of two experiments with standard error (*, p<0.05 and **, p<0.01).

Fig.7 Effect of oleamide on tyrosinase enzyme activity. (A) No significant change in tyrosinase enzyme activity was found when melanocytes alone were treated with oleamide. However, significant reduction in the tyrosinase enzyme activity was found when coculture of keratinocytes and melanocytes were treated with oleamide. (B) Addition of oleamide to the melanocyte lysate did not alter the tyrosinase enzyme activity indicating no direct inhibitory effect of oleamide on tyrosinase enzyme activity. However, when the known inhibitor of tyrosinase activity, the Kojic acid was added to the melanocyte lysate we observed significant reduction in the tyrosinase activity.*, p<0.05, **, p<0.01 in comparison with control group.

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