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.
A chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. lic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. ORF2 encodes the UDP-galactose-4-epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. ORF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3' end of ORF1, all of galE, and the 5' end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.
a-Melanocyte stimulating hormone (a-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of a-, b-and g-MSH correlated clinically with malignant melanoma development, but other studies suggest a-MSH acts to retard invasion. In the present study, we investigated the action of a-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. a-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kB transcription factor. However, A375-SM and C8161 cells did not respond to a-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three lines and MC-2R on HBL and A375-SM lines. Receptor binding identified a similar affinity for a-MSH for all three lines with the highest number of binding sites on HBL cells. Only the HBL melanoma line demonstrated a detectable cyclic adenosine monophosphate (cAMP) response to a-MSH, although all three lines responded to acute a-MSH addition ( þ (À)-N 6 -(2-phenylisopropyl)-adenosine (PIA)) with an elevation in intracellular calcium. The nonresponsive lines displayed MC-1R polymorphisms (C8161, Arg (wt) 151/Cys 151; A375-SM, homozygous Cys 151), whereas the HBL line was wild type. Stable transfection of the C8161 line with wild-type MC-1R produced cells whose invasion was significantly inhibited by a-MSH. From this data, we conclude that a-MSH can reduce melanoma cell invasion and protect cells against proinflammatory cytokine attack in cells with the wild-type receptor (HBL).
We have previously shown alpha-melanocyte stimulating hormone to protect melanocytes and melanoma cells from the proinflammatory actions of tumor necrosis factor-alpha. The aim of the study was to extend this work to look into the influence of tumor necrosis factor-alpha on melanoma cell attachment, invasion, and integrin expression and ask to what extent alpha-melanocyte stimulating hormone might protect cells from tumor necrosis factor-alpha stimulation of increased integrin expression. HBL human melanoma cells were studied under resting and stressed conditions using tumor necrosis factor-alpha as a proinflammatory cytokine. Functional information on the actions of tumor necrosis factor-alpha on melanoma cells was obtained by examining the strength of attachment of melanoma cells to substrates and the ability of melanoma cells to invade through fibronectin. alpha3, alpha4, and beta1 integrin expression was detected by Western immunoblotting and the ability of alpha-melanocyte stimulating hormone to oppose the actions of tumor necrosis factor-alpha was studied on HBL cell attachment, invasion, and integrin subunit expression. Our results show that tumor necrosis factor-alpha increases the number of melanoma cells attaching to collagen (types I and IV) and tissue culture polystyrene, increases ability to invade through fibronectin, and upregulates the expression of alpha3 (28%), alpha4 (90%), and beta1 (65%) integrin subunit expression. In contrast, alpha-melanocyte stimulating hormone reduced cell attachment, invasion, and integrin expression and opposed the stimulatory effects of tumor necrosis factor-alpha. In conclusion this study provides further evidence of alpha-melanocyte stimulating hormone acting to "protect" melanoma cells from proinflammatory cytokine action. Our data support a hypothesis that an inflammatory environment would promote melanoma invasion and that the anti-invasive actions of alpha-melanocyte stimulating hormone are consistent with its working in an anti-inflammatory capacity.
alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.
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