Inhibition of Tumor Necrosis Factor-α Stimulated NFκB/p65 in Human Keratinocytes by α-Melanocyte Stimulating Hormone and Adrenocorticotropic Hormone Peptides

Abstract

Alpha-melanocyte stimulating hormone (alpha-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-alpha in a number of tissues, including skin. We have previously shown that alpha-MSH can inhibit tumor necrosis factor-alpha stimulated intercellular adhesion molecule 1 upregulation and nuclear factor kappaB (NFkappaB) transcription factor activation in melanocyte and melanoma cells. It is thought, however, that this MSH biology may also extend to other cells of the skin and in this study we extend our work to keratinocytes. We have investigated in detail the ability of three alpha-MSH peptides to inhibit tumor necrosis factor alpha stimulated NFkappaB activation in nonpigmentary HaCaT keratinocytes (alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val) and two adrenocorticotropic hormone (ACTH) peptides (1-17 and 1-39), reported to be present in skin tissue. NFkappaB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor alpha stimulated NFkappaB activation, whereas ACTH 1-17 and 1-39 did not, in the HaCaT keratinocytes. MSH peptides and ACTH 1-39 were effective, however, at inhibiting NFkappaB activation in normal human keratinocytes. Immunolabeling of inhibitor kappaBalpha of NFkappaB (IkappaBalpha) revealed an abnormal localization to the nucleus of HaCaT cells, which was unaffected by MSH/ACTH peptides. In contrast, normal human keratinocytes showed a normal IkappaBalpha distribution that responded to MSH/ACTH with nuclear translocation. Our data support previous work on the role of MSH/ACTH peptides as immunomodulatory/anti-inflammatory regulators, and extend this work to keratinocytes identifying a novel IkappaBalpha mechanism and extends findings to ACTH peptides, identifying an abnormal IkappaBalpha mechanism in the immortal HaCaT versus normal keratinocyte.

Anti-inflammatory and anti-invasive effects of α-melanocyte-stimulating hormone in human melanoma cells

“…HEK293 cells (which do not express MC-1R) did not label, as confirmed previously (Moustafa et al, 2002). No immunoreactivity was observed for cells incubated with isotype control serum.…”

“…a-Melanocyte-simulating hormone can inhibit TNF-a-stimulated NF-kB activity in human melanocytes, melanoma cells and keratinocytes (Haycock et al, 1999(Haycock et al, , 2000Moustafa et al, 2002). In all of these cells, a-MSH inhibited the response to TNF-a by around 50%.…”

“…For the NFkB/p65 studies, melanoma cells were incubated in the appropriate medium (HMCM/ EMEM)7a-MSH (10 À12 10 À6 M) for 15 min, followed by a 1 h incubation in fresh medium7TNF-a (300 U ml À1 ). Cells were immunofluorescently labelled for MC-1R and NFkB/p65 as detailed previously (Moustafa et al, 2002). Melanocortin-1 receptors and NFkB/p65 in each melanoma were visualised using epifluorescent illumination (FITC filter, l ex ¼ 490 nm, l em ¼ 523 nm), and nuclei were visualised using a rhodamine filter (l ex ¼ 555 nm, l em ¼ 580 nm) with a Nikon X600 microscope.…”

“…Digital images of MC-1R/NFkB/p65 and nuclei were captured and overlaid using Adobe Photoshop. Cellular movement of NF-kB/p65 was evaluated as described previously (Moustafa et al, 2002).…”

“…Western blot analysis was carried out as detailed previously (Moustafa et al, 2002). Immunoreactive bands were visualized using an ECL kit.…”