Inhibition of Tumor Necrosis Factor-α Stimulated NFκB/p65 in Human Keratinocytes by α-Melanocyte Stimulating Hormone and Adrenocorticotropic Hormone Peptides
2002; Elsevier BV; Volume: 119; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2002.19602.x
ISSN1523-1747
AutoresManar Moustafa, E. Helen Kemp, Sheila MacNeil, Marika Szabo, John W. Haycock, Ghanem E. Ghanem, Renato Morandini,
Tópico(s)NF-κB Signaling Pathways
Resumoα-Melanocyte stimulating hormone (α-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-α in a number of tissues, including skin. We have previously shown that α-MSH can inhibit tumor necrosis factor-α stimulated intercellular adhesion molecule 1 upregulation and nuclear factor κB (NFκB) 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 α-MSH peptides to inhibit tumor necrosis factor α stimulated NFκB activation in nonpigmentary HaCaT keratinocytes (α-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. NFκB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. α-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor α stimulated NFκB 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 NFκB activation in normal human keratinocytes. Immunolabeling of inhibitor κBα of NFκB (IκBα) 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 IκBα 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 IκBα mechanism and extends findings to ACTH peptides, identifying an abnormal IκBα mechanism in the immortal HaCaT versus normal keratinocyte. α-Melanocyte stimulating hormone (α-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-α in a number of tissues, including skin. We have previously shown that α-MSH can inhibit tumor necrosis factor-α stimulated intercellular adhesion molecule 1 upregulation and nuclear factor κB (NFκB) 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 α-MSH peptides to inhibit tumor necrosis factor α stimulated NFκB activation in nonpigmentary HaCaT keratinocytes (α-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. NFκB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. α-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor α stimulated NFκB 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 NFκB activation in normal human keratinocytes. Immunolabeling of inhibitor κBα of NFκB (IκBα) 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 IκBα 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 IκBα mechanism and extends findings to ACTH peptides, identifying an abnormal IκBα mechanism in the immortal HaCaT versus normal keratinocyte. adrenocorticotropic hormone α-melanocyte stimulating hormone electrophoretic mobility shift assay human embryonal kidney inhibitor κBα of NFκB inositol trisphosphate melanocortin melanocortin receptor nuclear factor κB proopiomelanocortin Tris-buffered saline Tween α-Melanocyte stimulating hormone (α-MSH) is a 13 amino acid peptide that arises by proteolytic cleavage of the proopiomelanocortin (POMC) pro-hormone precursor molecule. The peptide is produced in a number of tissues including the intermediate lobe of the pituitary, gut, and skin (Eberle, 1988Eberle A.N. The Melanotrophins: Chemistry, Physiology and Mechanism of Action. Karger, Basel1988Google Scholar). α-MSH has many biologic effects, most notably on eumelanogenic pigmentation of the skin (Eberle and Cone, 2000Eberle A.N. Proopiomelanocortin and the melanocortin peptides.in: Cone R.D. Melanocortin Receptors. Humana Press, Totowa, NJ2000: 3-68Google Scholar). An increasing literature also documents other extra-pigmentary roles, including inhibition of fever, inflammation, and immunomodulation (Hiltz and Lipton, 1989Hiltz M.E. Lipton J.M. Antiinflammatory activity of a COOH-terminal fragment of the neuropeptide α-MSH.FASEB J. 1989; 3: 2282-2284Crossref PubMed Scopus (74) Google Scholar;Hiltz et al., 1991Hiltz M.E. Catania A. Lipton J.M. Anti-inflammatory activity of α-MSH (11–13) analogs: Influences of alteration in stereochemistry.Peptides. 1991; 12: 767-771Crossref PubMed Scopus (44) Google Scholar;Lipton et al., 1991Lipton J.M. Macaluso A. Hiltz M.E. Catania A. Central administration of the peptide α-MSH inhibits inflammation in the skin.Peptides. 1991; 12: 795-798Crossref PubMed Scopus (84) Google Scholar;Lipton et al., 1998Lipton J.M. Catania A. Zhao H. α-MSH-induced modulation of inflammation: Lo.Exp Dermatol. 1998; 7: 226Google Scholar;Martin et al., 1991Martin L.W. Catania A. Hiltz M.E. Lipton J.M. Neuropeptide α-MSH antagonises IL-6 and TNFinduced fever.Peptides. 1991; 12: 297-299Crossref PubMed Scopus (72) Google Scholar;Catania and Lipton, 1993Catania A. Lipton J.M. Alpha-melanocyte stimulating hormone in the modulation of host reactions.Endocrine Rev. 1993; 14: 564-576PubMed Google Scholar;Watanabe et al., 1993Watanabe T. Hiltz M.E. Catania A. Lipton J.M. Inhibition of IL-1β-induced peripheral inflammation by peripheral and central administration of analogs of the neuropeptide α-MSH.Brain Res Bull. 1993; 32: 311-314Crossref PubMed Scopus (54) Google Scholar;Ceriani et al., 1994Ceriani G. Macaluso A. Catania A. Lipton J.M. Central neurogenic antiinflammatory action of α-MSH. Modulation of peripheral inflammation induced by cytokines and other mediators of inflammation.Neuroendcrinology. 1994; 59: 138-143Crossref PubMed Scopus (84) Google Scholar;Lipton and Catania, 1997Lipton J.M. Catania A. Anti-inflammatory actions of the neuroimmunomodulator α-MSH.Immunol Today. 1997; 18: 140-145Abstract Full Text PDF PubMed Scopus (369) Google Scholar;Rajora et al., 1997Rajora N. Boccoli G. Burns D. Sharma S. Catania A.P. Lipton J.M. α-MSH modulates local and circulating tumor necrosis factor-α in experimental brain inflammation.J Neurosci. 1997; 17: 2181-2186Crossref PubMed Google Scholar;Ichiyama et al., 1999bIchiyama T. Zhao H. Catania A. Furukawa S. Lipton J.M. α-Melanocyte-stimulating hormone inhibits NFkB activation and IkBα degradation in human glioma cells and in experimental brain inflammation.Exp Neurol. 1999; 157: 359-365Crossref PubMed Scopus (83) Google Scholar;Taherzadeh et al., 1999Taherzadeh S. Sharma S. Chhajlani V. et al.α-MSH and its receptors in regulation of tumor necrosis factor-α production by human monocyte/macrophages.Am J Physiol. 1999; 276: 1289-R1294PubMed Google Scholar). In human skin, α-MSH is immunoreactive in keratinocytes and melanocytes and studies have previously suggested a regulatory involvement in melanocyte pigmentation and proliferation (Hedley et al., 1998aHedley S.J. Gawkrodger D.J. Weetman A.P. MacNeil S. α-MSH and melanogenesis in normal human adult melanocytes.Pigment Cell Res. 1998; 11: 45-56Crossref PubMed Scopus (43) Google Scholar). α-MSH signaling is transmitted via a family of specific melanocortin (MC) G-protein linked receptors. Five MC receptors have been cloned to date (MC-1R to MC-5R) and in skin MC-1R is expressed on cutaneous melanocytes and melanoma cells (Chhajlani and Wikberg, 1992Chhajlani V. Wikberg J.E. Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.FEBS Lett. 1992; 309: 417-420Abstract Full Text PDF PubMed Scopus (577) Google Scholar;Mountjoy et al., 1992Mountjoy K.G. Robbins L.S. Mortrud M.T. Cone R.D. The cloning of a family of genes that encode the melanocortin receptors.Science. 1992; 257: 1248-1251Crossref PubMed Scopus (1457) Google Scholar). MC-1R, however, is also expressed on other cell types including keratinocytes, endothelial cells, monocytes, and neural cells (Bhardwaj et al., 1997Bhardwaj R.S. Becher E. Mahnke K. Hartmeyer M. Schwarz T. Scholtzen T. Luger T.A. Evidence for the differential expression of the functional α melanocyte stimulating hormone receptor MC-1 on human monocytes.J Immunol. 1997; 158: 3378-3384PubMed Google Scholar;Hartmeyer et al., 1997Hartmeyer M. Scholzen T. Becher E. Bhardwaj R.S. Fastrich M. Schwarz T. Luger T.A. Human microvascular endothelial cells (HMEC-1) express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with αMSH.J Immunol. 1997; 159: 1930-1937PubMed Google Scholar). The mechanism by which α-MSH acts as an anti-inflammatory peptide is not completely resolved. It is known to inhibit proinflammatory cytokine production/action e.g., tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β); Weiss et al., 1991Weiss J.M. Sundar S.K. Cierpial M.A. Ritchie J.C. Effects of interleukin-1 infused into brain are antagonised by α-MSH in a dose-dependent manner.Eur J Pharmacol. 1991; 192: 177-179Crossref PubMed Scopus (50) Google Scholar and to increase anti-inflammatory cytokine production (e.g., IL-10;Bhardwaj et al., 1996Bhardwaj R.S. Shwarz A. Becher E. Mahnke K. Aragane Y. Schwarz T. Luger T.A. Pro-opiomelanocortin-derived peptides induce IL-10 production in human monocytes.J Immunol. 1996; 156: 2517-2521PubMed Google Scholar). We have previously reported α-MSH to have a role in cutaneous immunomodulation, showing an ability to inhibit TNFα upregulation of surface intercellular adhesion molecular 1 (ICAM-1) expression (prerequisite for T lymphocyte binding) in melanocytes and melanoma cells (Hedley et al., 1998bHedley S.J. Gawkrodger D.J. Weetman A.P. Morandini R. Boeynaems J.-M. Ghanem G. MacNeil S. α-melanocyte stimulating hormone inhibits tumour necrosis factor-α stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines.Br J Dermatol. 1998; 138: 536-543Crossref PubMed Scopus (41) Google Scholar;Morandini et al., 1998Morandini R. Boeynams J.M. Hedley S.J. MacNeil S. Ghanem G. Modulation of ICAM-1 expression by α-MSH in human melanoma cells and melanocytes.J Cell Physiol. 1998; 175: 276-282Crossref PubMed Scopus (50) Google Scholar). We have also demonstrated that α-MSH inhibits the TNFα activation of the nuclear factor κB (NFκB) transcription factor in ocular and cutaneous melanocytes and melanoma cells (Haycock et al., 1999aHaycock J.W. Wagner M. Morandini R. Ghanem G. Rennie I.G. MacNeil S. α-Melanocyte-stimulating hormone inhibits NFkB activation in human melanocytes and melanoma cells.J Invest Dermatol. 1999; 113: 560-566Crossref PubMed Scopus (66) Google Scholar;Haycock et al., 1999bHaycock J.W. Wagner M. Morandini R. Ghanem G. Rennie I.G. MacNeil S. α-MSH immunomodulation acts via Rel/NFκB in cutanous and ocular melanocytes and in melanoma cells.Ann NY Acad Sci. 1999; 885: 396-399Crossref PubMed Scopus (18) Google Scholar). Acute NFκB activation is responsible for expression of several inflammatory and immune system genes, and hence inhibition by α-MSH is one signaling pathway by which this peptide may exert anti-inflammatory control. Furthermore, we have also demonstrated that α-MSH (and peptide derivatives) decrease acute intracellular generation of reactive oxygen species, directly detectable as peroxide species (and indirectly as perturbation of glutathione peroxidase activity), in both a melanoma and a keratinocyte cell line (HaCaT) (Haycock et al., 2000Haycock J.W. Rowe S.J. Cartledge S. et al.α-Melanocyte-stimulating hormone reduces impact of proinflammatory cytokine and peroxide-generated oxidative stress on keratinocytes and melanoma cell lines.J Biol Chem. 2000; 275: 15629-15636Crossref PubMed Scopus (89) Google Scholar). Peroxide species are reported to be a mechanism by which NFκB is rapidly activated, and therefore inhibition by the MSH peptides may explain signaling control over NFκB activation and ICAM-1 expression. An MSH attenuation of the ICAM-1 response to TNFα would be consistent with protection of cells from the immune system, and we have recently shown that α-MSH reduces the ability of melanoma cells to bind to T lymphocytes (Hedley et al., 2000Hedley S.J. Murray A. Sisley K. Ghanem G. Morandini R. Gawkrodger D.J. MacNeil S. α-Melanocyte stimulating hormone can reduce T–cell interaction with melanoma cells in vitro.Melanoma Res. 2000; 10: 323-330Crossref PubMed Scopus (14) Google Scholar). In addition to the native α-MSH (1–13) peptide, several MSH peptide derivatives are reported to possess anti-inflammatory biology. The most notable of these is the carboxyl-terminal tripeptide MSH 11–13 (Lys-Pro-Val). Indeed it is thought that this is the minimum obligatory MSH sequence needed to demonstrate an antiinflammatory effect in vivo (Hiltz and Lipton, 1989Hiltz M.E. Lipton J.M. Antiinflammatory activity of a COOH-terminal fragment of the neuropeptide α-MSH.FASEB J. 1989; 3: 2282-2284Crossref PubMed Scopus (74) Google Scholar;Hiltz et al., 1991Hiltz M.E. Catania A. Lipton J.M. Anti-inflammatory activity of α-MSH (11–13) analogs: Influences of alteration in stereochemistry.Peptides. 1991; 12: 767-771Crossref PubMed Scopus (44) Google Scholar). A number of previous studies report on the MSH responsiveness of pigmentary skin cells; however, it is known that the immunoregulatory biology displayed by α-MSH is reported for a number of dissimilar tissues, and so it is feasible that the anti-inflammatory biology of the MSH peptides with respect to skin is not restricted solely to melanocytes and melanoma cells. Furthermore, it has recently been reported that adrenocorticotropic hormone (ACTH) and a truncated peptide (ACTH 1–17) are present in skin tissue and that ACTH 1–17 binds to the MC-1R (Tsatmali et al., 1999Tsatmali M. Yukitake J. Thody A.J. ACTH 1–17 is a more potent agonist at the human MC1 receptor than α-MSH.Cell Mol Biol. 1999; 45: 1029-1034PubMed Google Scholar). Therefore in this study we investigated MC-1R and MC-2R expression in the human HaCaT keratinocyte cell line and in normal human keratinocytes and investigated whether MSH/ACTH peptides were able to influence the response of cells to proinflammatory cytokine stimulation assessed by an ability to inhibit TNFα-stimulated NFκB/p65 activity. The HaCaT human keratinocyte cell line was kindly supplied by Professor N.E. Fusenig (Institute of Biochemistry, German Cancer Research Center, Heidelberg, Germany). Cells were grown in 24-well plates in Dulbecco's modified Eagle's medium (Sigma, Poole, U.K.) supplemented with 5% fetal bovine serum (Labtech International, U.K.), 2 mM L-glutamine (Sigma), 100 U penicillin per ml, and 100 μg streptomycin per ml (Gibco, Paisley, U.K.). Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. Human embryonic kidney (HEK293) cells were kindly supplied by Professor R.J. Ross (Section of Medicine, Division of Clinical Sciences, University of Sheffield, U.K.). Cells were grown in 24-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U penicillin per ml, and 100 μg streptomycin per ml. Normal primary human keratinocytes were established from full-thickness skin obtained from abdominoplasty operations as described previously (Goberdan et al., 1993Goberdan N.J. Dawson R.A. Freedlander E. MacNeil S. A calmodulin-like protein as an extracellular mitogen for the keratinocyte.Br J Dermatol. 1993; 129: 678-688Crossref PubMed Scopus (32) Google Scholar). Briefly, keratinocytes were harvested from skin tissue pretreated with trypsin–ethylenediamine tetraacetic acid (EDTA) (18 h) and cultured with Defined Keratinocyte Medium (SFM, Gibco, U.K.) on type I rat tail collagen at 31 μl per cm2. First passage keratinocytes were used for experimentation. Cells were kept proliferative and experimentation started when cells reached 60% confluence. All peptides were from Bachem (Bubendorf, Switzerland): α-MSH, MSH 11–13 (L-K-L-P-L-V), MSH 11–13 (L-K-L-P-D-V), ACTH 1–17, or ACTH 1–39 (10–7 M to 10–13 M) were added to cells for an incubation period of 15 min followed by TNFα (Boehringer-Mannheim, Lewes, U.K.) addition (2.0 ng per ml). Cells were incubated for a further 60 min, medium removed, and washed with phosphate-buffered saline (PBS) three times, fixed with 2% paraformaldehyde (BDH Chemicals, Poole, U.K.) for 10 min, washed again with PBS (×3) and permeabilized using 0.1%Triton X100 (BDH) for 20 min, washed with PBS (×3), and then neutralized with 50 mM ammonium chloride (BDH) in PBS for 10 min. Cells were permeablized for immuno-labeling of NFκB/p65 or IκBα with Triton-X100 (0.1% wt/vol). Non-permeablized cells were used for MC-1R or MC-2R immunolabeling. After permeabilization, samples were washed with PBS (×3) and nonspecific protein sites were blocked using 5% dried-milk powder (in PBS, 1 h). Cells were then incubated with the appropriate primary antibody: (a) anti-NFκB/p65 (C-20) goat polyclonal IgG antibody (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (b) anti-IκB-α (C-21) rabbit polyclonal IgG antibody (sc-371) for 1 h at room temperature and pressure (1:100 vol/vol, in PBS); (c) anti-MC-1R (N-19) goat polyclonal IgG antibody (sc-6875) for 18 h at 4°C (1:50 vol/vol, in blocking buffer/PBS); or (d) anti-MC-2R (C-16) goat polyclonal IgG (sc-6876) for 18 h at 4°C (1:50 vol/vol, in blocking buffer/PBS). Cells were washed with PBS (×3) and then incubated with biotinylated anti-goat IgG antibody (BA-5000, Vector Laboratories, CA, 1:1000 vol/vol in PBS) or biotinylated anti-rabbit IgG antibody (BA-1000, Vector Laboratories) for 1 h at room temperature and pressure, washed with PBS (×3), followed by incubation with fluorescein streptavidin (SA-5001, Vector Laboratories) 1:100 vol/vol in PBS for 30 min. Cover slips were mounted using Vectashield mounting medium (H-1300, Vector Laboratories) containing propidium iodide to label nuclei (at 1.5 μg per μl). NFκB/p65 in normal human keratinocytes and HaCaT cells were observed visually using epifluorescent microscopy with differential Pinkel filter sets for fluorescein (NFκB/p65: λex=494 nm, λem=518 nm) and propidium iodide (nuclei: λex=536 nm, λem=617 nm). HaCaT keratinocytes, normal human keratinocytes, and HEK293 cells were seeded into six-well plates at 105 cells per well and grown until 60% confluent. Total cellular protein was then extracted for protein measurement (assessed using bicinchoninic acid assay kit; Pierce, Rochford, IL) and the remainder of the extract was incubated at 100°C for 2 min in a 75 mM Tris–HCl buffer pH 6.8 (containing 15% sodium dodecyl sulfate, 5%β-mercaptoethanol, 20% glycerol, 0.001% bromophenol blue). Fifteen micrograms of total protein was loaded per gel track and samples were electrophoresed according to the method of Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207129) Google Scholar (Mini Protean II dual slab cell, Bio-Rad, Hemel Hempstead, UK) using a 5% stacking gel and 10% resolving gel for 1 h (200 V, constant voltage). Proteins were transferred onto 0.45 μm nitrocellulose membrane (according to the method of Towbin et al., 1979Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci (USA). 1979; 76: 4350-4354Crossref PubMed Scopus (44911) Google Scholar; using a Mini Trans-Blot system, Bio-Rad; 100 V, constant voltage). MC-1R and MC-2R immunoreactive bands were visualized as follows: unreacted binding sites were blocked with 5% (wt/vol) commercial dried milk powder in TBST (10 mM Tris–HCl, 0.15 M NaCl, 0.05% (wt/vol) Tween 20, pH 8.0), followed by incubation with primary rabbit polyclonal IgG specific antibody to MC-1R (N-19, sc-6875) or MC-2R (C-16, sc-6876) (Santa Cruz Biotechnology; diluted 1:50 vol/vol with 5% commercial dried milk powder in TBST) for 18 h at 4°C, washed with TBST (3×10 min), and incubated with horseradish-peroxidase-conjugated goat antirabbit secondary antibody diluted 1:2000 (vol/vol) in TBST (Dako, Cambridge, U.K.) for 1 h at room temperature. Immunoreactive bands were visualized using 3,3′-diaminobenzidine (0.4 μg per ml)/hydrogen peroxide (0.7 μl per ml). Prestained electrophoresis molecular weight standard markers were run in parallel for calibration purposes (SDS-7B, Sigma). HEK293 cells were transiently transfected by the Calcium Phosphate Transfection System (Gibco) using a pRc/CMV-MC-1R plasmid (kindly donated by Professor J.E.S. Wikberg, Uppsala University, Sweden). Cells were cotransfected with a β-galactosidase plasmid for calculating transfection efficiency. After transfection, cells were cultured for a further 3 d and processed for Western immunoblotting. Normal human keratinocytes and HaCaT keratinocytes were checked for their ability to bind α-MSH. Binding assays were performed using the following modification of a previously published method (Ghanem et al., 1988Ghanem G.E. Comunale G. Libert A. Vercammen-Grandjean A. Lejeune F.J. Evidence for alpha-melanocyte-stimulating hormone (α-MSH) receptors on human malignant melanoma cells.Int J Cancer. 1988; 41: 248-255Crossref PubMed Scopus (104) Google Scholar). Briefly, 250×103 dpm of 125I-[Nle4, Dphe7]-α-MSH in 100 μl was added to 106 cells (100 μl) previously mixed with [Nle4, Dphe7]-α-MSH (concentration ranging from 10-13 to 10-6 M) in PBS pH 7.2 containing 0.1% bovine serum albumin, 6.25 mM Hepes, and 0.1% trasylol. After mixing, the tubes were incubated at 37°C for 45 min before cell separation by repeated centrifugation and washing. The pellet-associated radioactivity was then measured in a gamma counter. Following the above protocol for HaCaT cell treatment with TNFα and α-MSH combinations, cells were harvested using a scraper into 10 mM Hepes buffer (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, and 0.1% Nonidet p-40. Nuclear extracts were prepared as described previously (Dignam et al., 1983Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucl Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9154) Google Scholar). Briefly, samples were centrifuged (12,000×g, 5 min) and the pellet was suspended in 20 mM Hepes buffer, pH 7.9, containing 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, incubated on ice (10 min), and centrifuged (12,000×g, 5 min). Supernatant was diluted in 20 mM Hepes buffer, with 0.05 M KCl, 0.2 mM EDTA, 20% glycerol, and centrifuged (12,000×g, 5 min). Supernatant from the latter centrifugation (containing the nuclear fraction) was measured for protein content using the bicinchoninic acid technique (Pierce). Five micrograms of protein from each sample were mixed with a reaction buffer (1 mM EDTA, 50 mM NaCl, 10 mM Tris) containing 50 μg per ml poly(dI-dC) (Promega, Southampton, U.K.) and end-labeled oligonucleotide probe. The NFκB probe was a 25-mer double-stranded synthetic oligonucleotide (VH Bio, Gosforth, U.K.) containing the κB motif (5′-TGACAGAGGGGACTTCCGAGAGGA-3′). End-labeling was by T4 polynucleotide kinase (Promega) reaction with [γ-32P] ATP (NEN, Brussels, Belgium). Samples were loaded onto a 4% polyacrylamide gel (38:1) in 1×TBE (prerun for 30 min) and run at 100 V for 1 h, at 4°C. Gels were dried and exposed to film (Amersham hyperfilm) for 18 h at –70°C with intensifying screens (Hypercassette, Amersham, Little Chalfunt, UK). Quantification of NFκB activity was by scanning densitometry of the p50/p65 specific gel shift band (as a ratio of the excess unbound radio labeled oligonucleotide band, as internal control) using a GS-700 imaging densitometer in transmittance mode integrating optical density and cross-sectional area, using Molecular Analyst software (600 dpi, Bio-Rad). Cells were treated as described above, stimulated with or without TNFα (2.0 ng per ml) for 60 min, and then the nuclear fraction was extracted. An excess of cold native oligonucleotide (100 M) was added in addition to radiolabeled oligonucleotide in the sample binding reaction described above, prior to running samples as before. In addition, an excess of cold mutant oligonucleotide was also used (containing two base pair mutations (GA→CT) of the native oligonucleotide probe: 5′-TGACA-GAGGGCTCTTCCGAGAGGA-3′). For identification of subunit composition, nuclear extracts were incubated with rabbit polyclonal antibodies (Santa Cruz Biotechnology) against the p65 or p50 NFκB subunits. Student's paired t test was used to analyze data for identical cellular treatments between control untreated and treated samples. Positive immunolabeling for the human MC-1R was identified on the surface of nonpermeabilized normal human keratinocytes (Figure 1a). Positive immunolabeling was also identified for the MC-2R (Figure 1b). For both MC-1R and MC-2R we observed the immunolabeling position to be similar, exhibiting an even distribution over the surface membrane of each cell under high magification (×100, oil immersion objective lens). Captured photographic images give the impression of a "perinuclear" concentration. This observation can also be seen by eye and is more so due to the very small focal depth described by the microscope optics rather than a true description of receptor distribution. Alteration of the focal plane readily revealed membrane immunolabeling immediately above the nucleus position for both MC-1R and MC-2R. No detection was observed when primary anti-MC-R antibodies were substituted with isotype IgG immunosera (Figure 1c, micrograph illustrates counter labeling of nuclei for orientation purposes with an absence of MC-R labeling). HaCaT keratinocytes were also observed to label positively for MC-1R, exhibiting a similar punctate labeling appearance evenly distributed across the membrane surface (Figure 1d). In contrast to normal keratinocytes, a weaker positive labeling for MC-2R was identified (Figure 1e). Minor differences in labeling intensity are interpreted with some caution in relation to relative levels of receptor expression; however, we would suggest that if HaCaT keratinocytes express MC-2R the level of expression is very low. These cells did not label when isotype IgG antisera were used in place of primary anti-MC-R (Figure 1f, micrograph counterlabeling of nuclei alone illustrated). Non-transfected HEK293 cells were used as a negative control, as these cells are reported not to express the MC-1R (Tsatmali et al., 1999Tsatmali M. Yukitake J. Thody A.J. ACTH 1–17 is a more potent agonist at the human MC1 receptor than α-MSH.Cell Mol Biol. 1999; 45: 1029-1034PubMed Google Scholar). It was observed, however, that HEK293 cells exhibited a degree of positive MC-1R immunolabeling (Figure 1g). In comparison, the normal human keratinocytes and HaCaT keratinocytes exhibited substantially higher immunolabeling intensity (Figure 1a, Figure 1dversusFigure 1g). This suggests that either the primary antibody used exhibits a degree of cross-reactivity, or that a small degree of MC-1R expression arises in this cell type. It was noted that very little immunolabeling was present for the MC-2R of HEK293 cells (Figure 1h, counterlabeling for nuclei alone shown). Furthermore, negative labeling was observed when isotype IgG serum was used in place of a primary antibody (Figure 1i, counterlabeling for nuclei alone shown). Western immunoblotting of normal human keratinocytes (Figure 2, lane 3) and HaCaT keratinocyes (Figure 2, lane 4) for the MC-1R identified a single immunoreactive product at a molecular weight of 35 kDa (indicated by arrow adjacent to lane 4). This corresponds to the predicted molecular size for the MC-1R. In addition, Western blots labeled for the MC-2R revealed a major immunoreactive band observed for both normal human keratinocytes (Figure 2, lane 5) and HaCaT keratinocytes (Figure 2, lane 6) at 39 kDa, corresponding closely to the predicted molecular weight of 34 kDa for the MC-2R. To confirm immunoreactive specificity of Western blotting for the MC-1R, nontransfected HEK293 cell sample extracts were immunolabeled in parallel with HEK293 cells that were transiently transfected with a pRc/CMV-MC-1R expression vector. We observed that nontransfected cells did immunolabel very faintly for a 35 kDa product (Figure 2, lane 1), suggesting that HEK293 cells may actually express very low levels of MC-1R protein (somewhat supportive of immunofluorescent microscopy data); however, MC-1R transiently transfected cells demonstrated an identical size immunoreactive product, but with a very much higher band labeling intensity (Figure 2, lane 2; all sample lanes contained 15 μg total cellular protein loaded per gel track). It is worth noting that higher molecular weight bands were identified by the anti-MC-1R antibody, suggestive of the possibility of cross-reactivity with non-MC-1R protein. The HaCaT keratinocytes demonstrated a specific ability to bin
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