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Abundant Expression of Vasoactive Intestinal Polypeptide Receptor VPAC2 mRNA in Human Skin

2001; Elsevier BV; Volume: 117; Issue: 3 Linguagem: Inglês

10.1046/j.0022-202x.2001.01449.x

1523-1747

Tanja C. Fischer, Petra Hartmann, Christoph Löser, Jochen Springer, Christian Peiser, Q. Thai Dinh, Axel Fischer, David A. Groneberg,

Receptor Mechanisms and Signaling

To the Editor: Vasoactive intestinal polypeptide (VIP) is a bioactive peptide that influences many aspects of cell function and differentiation. The 28-amino acid polypeptide belongs to the glucagon/secretin superfamily and is widely expressed in the central nervous system and in peripheral tissues including lung and skin, where it has been shown to have a multitude of biological functions (Dickinson and Fleetwood-Walker, 1999Dickinson T. Fleetwood-Walker S.M. VIP & PACAP. very important in pain.Trends Pharmacol Sci. 1999; 20: 324-329Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). VIP is abundantly present in cutaneous autonomic and sensory nerve fibers (Eedy et al., 1994Eedy D.J. Shaw C. Johnston C.F. Buchanan K.D. The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography.Clin Exp Dermatol. 1994; 19: 463-472Crossref PubMed Scopus (38) Google Scholar) where it acts as neuromodulator and participates in the regulation of regional blood flow. As the expression and localization of VPAC2 mRNA has not been examined so far, this study was carried out to correlate VIP and its receptor in the human skin. Surgically resected human skin samples (n = 24) were obtained from surplus remnants in excess of that required for pathologic examination after informed consent. For mRNA in situ hybridization, VPAC2 receptor cRNA probes were generated by a standardized protocol using the sequence corresponding to the region spanning from TM 3–7 of the human VPAC2 receptor from TSUP1 human T lymphoblast cDNA which was subcloned into a specific vector (pGEM-T vector, Promega, Madison, WI) (Groneberg et al., 2001aGroneberg D.A. Hartmann P. Dinh Q.T. Fischer A. Expression and distribution of vasoactive intestinal polypeptide receptor VPAC2 mRNA in human airways.Lab Invest. 2001; 81: 749-755Crossref PubMed Scopus (62) Google Scholar). For antisense probes, plasmid linearization by restriction with Spe I was followed by transcription with T7 polymerase, for sense probes, the Nco I linearized plasmids were transcribed with SP6 polymerase (all Roche Diagnostics, Mannheim, Germany). The probes were checked for their integrity by TAE-agarose gel electrophoresis and ethidium bromide staining. The VPAC2 receptor mRNA distribution was assessed by nonisotopic in situ hybridization using a standardized protocol (Groneberg et al., 2001bGroneberg D.A. Nickolaus M. Springer J. et al.Localization of the peptide transporter PEPT2 in the Lung: implications for pulmonary oligopeptide uptake.Am J Pathol. 2001; 158: 707-714Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) with 6–8 µm cryostat sections of 4% paraformaldehyde fixed biopsies. High resolution interference contrast microscopy revealed abundant staining in all areas of the biopsies Figure 1. Positive VPAC2 mRNA-signals were localized to the cytoplasm of keratinocytes with a signal intensity that was maximal at the basal zone and decreasing to superficial layers Figure 1a. In the deep part of the dermis, VPAC2 mRNA hybridization signals were present in cells of eccrine sweat glands and in cells of the germinative epithelium, matrix, and medulla of the hair follicle Figure 1c. Also, positive VPAC2 mRNA hybridization signals were localized to endothelial and mononuclear immune cells. Control hybridizations on alternate sections with equivalent amounts of the digoxigenin labeled sense-probe using the same hybridization conditions and washing stringencies were unstained Figure 1b, d. To relate the distribution of VPAC2 mRNA to its ligand VIP, immunohistochemical studies were carried out using mouse polyclonal (1:1000, Biogenesis, Poole, U.K.) or monoclonal (1:100 Charles River, Southbridge, MA) VIP-antibodies as described before (Fischer et al., 1998Fischer A. Canning B.J. Undem B.J. Kummer W. Evidence for an esophageal origin of VIP-IR and NO synthase-IR nerves innervating the guinea pig trachealis: a retrograde neuronal tracing and immunohistochemical analysis.J Comp Neurol. 1998; 394: 326-334Crossref PubMed Scopus (50) Google Scholar) with 6–8 µm cryostat sections and antimouse fluorescein-5-isocyanate antiserum (1:400, Amersham, Braunschweig, Germany) and anti-rabbit biotin (1:200) – strepatavidin Texas Red (1:400, Amersham) as secondary antibodies. Fluorescence microscopy demonstrated an abundant expression of VIP-immunoreactivity in nerve fibers which were found as branching networks surrounding blood vessels, eccrine sweat glands, and hair follicles Figure 1e and f and in direct contact to VPAC2 mRNA positive cells. Controls by omitting the primary or secondary antibodies and incubation with the preimmune serum did not reveal specific immunosignals. VIP binding sites have so far been shown in sweat glands using unspecific binding techniques (Heinz-Erian et al., 1986Heinz-Erian P. Paul S. Said S.I. Receptors for vasoactive intestinal peptide on isolated human sweat glands.General Pharmacol. 1986; 17: 321-326Crossref PubMed Scopus (21) Google Scholar) and in allergic contact dermatitis (Lundeberg and Nordlind, 1999Lundeberg L. Nordlind K. Vasoactive intestinal polypeptide in allergic contact dermatitis. an immunohistochemical and radioimmunoassay study.Arch Dermatol Res. 1999; 291: 201-206https://doi.org/10.1007/s004030050394Crossref PubMed Scopus (19) Google Scholar). There is also a large body of evidence on the functional role of VIP as a possible sensory neuropeptide in normal skin and dermatologic disorders. VIP protein expression has been demonstrated in normal skin of young (Eedy et al., 1994Eedy D.J. Shaw C. Johnston C.F. Buchanan K.D. The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography.Clin Exp Dermatol. 1994; 19: 463-472Crossref PubMed Scopus (38) Google Scholar) and elderly (Abdel-Rahman et al., 1992Abdel-Rahman T.A. Collins K.J. Cowen T. Rustin M. Immunohistochemical, morphological and functional changes in the peripheral sudomotor neuro-effector system in elderly people.J Auton Nerv Syst. 1992; 37: 187-197Abstract Full Text PDF PubMed Scopus (44) Google Scholar) people and in dermatologic disorders such as nodular prurigo (Abadia Molina et al., 1992Abadia Molina F. Burrows N.P. Jones R.R. Terenghi G. Polak J.M. Increased sensory neuropeptides in nodular prurigo: a quantitative immunohistochemical analysis.Br J Dermatol. 1992; 127: 344-351Crossref PubMed Scopus (109) Google Scholar) or allergic contact dermatitis (Lundeberg and Nordlind, 1999Lundeberg L. Nordlind K. Vasoactive intestinal polypeptide in allergic contact dermatitis. an immunohistochemical and radioimmunoassay study.Arch Dermatol Res. 1999; 291: 201-206https://doi.org/10.1007/s004030050394Crossref PubMed Scopus (19) Google Scholar). Also the functional role of VIP in the normal skin and in disorders like contact dermatitis (Bondesson et al., 1996Bondesson L. Nordlind K. Mutt V. Liden S. Vasoactive intestinal polypeptide inhibits the established allergic contact dermatitis in humans.Ann N Y Acad Sci. 1996; 805: 702-707Crossref PubMed Scopus (9) Google Scholar) was analyzed; however, a detailed study of the mRNA expression of molecular distinct VIP receptors has not been carried out. As the molecular properties of the VIP receptors were identified in the past years, this study was designed to localize the mRNA of the inducible VIP receptor VPAC2 in normal human skin biopsies and resulted in abundant staining for VPAC2 mRNA in keratinocytes. Effects of VIP on keratinocyte cultures have been demonstrated earlier in vitro (Haegerstrand et al., 1989Haegerstrand A. Jonzon B. Dalsgaard C.J. Nilsson J. Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes.Proc Natl Acad Sci USA. 1989; 86: 5993-5996Crossref PubMed Scopus (157) Google Scholar). This demonstration of VPAC2 receptor mRNA in human keratinocytes in situ supports the previous in vitro findings and provides evidence that the mode of VIP modulation of cell proliferation in keratinocytes is mediated at least partly via the VPAC2 receptor. This finding is also supported by the recent demonstration of VPAC2 immunoreactivity in allergic contact dermatitis (Lundeberg and Nordlind, 1999Lundeberg L. Nordlind K. Vasoactive intestinal polypeptide in allergic contact dermatitis. an immunohistochemical and radioimmunoassay study.Arch Dermatol Res. 1999; 291: 201-206https://doi.org/10.1007/s004030050394Crossref PubMed Scopus (19) Google Scholar). A dense network of VIP-IR nerve fibers around sweat glands and hair follicles was shown in this study, and VIP has been reported to induce cAMP generation in human sweat glands (Tainio, 1987Tainio H. Cytochemical localization of VIP-stimulated adenylate cyclase activity in human sweat glands.Dev Biol. 1987; 123: 179-190Crossref PubMed Scopus (71) Google Scholar). By directly localizing VPAC2 mRNA to eccrine gland and hair follicle cells we provide first evidence for the identity of the VIP receptor subtype that is involved in VIP-induced upregulation of eccrine sweat secretion. VIP has been shown to be a potent relaxant of vascular smooth muscle (Lundberg et al., 1981Lundberg J.M. Anggard A. Emson P. Fahrenkrug J. Hokfelt T. Vasoactive intestinal polypeptide and cholinergic mechanisms in cat nasal mucosa: studies on choline acetyltransferase and release of vasoactive intestinal polypeptide.Proc Natl Acad Sci USA. 1981; 78: 5255-5259Crossref PubMed Scopus (115) Google Scholar). As VPAC2 receptor mRNA was not present in the vascular smooth muscle layer of cutaneous vessels, our results indicate that VIP-induced dermal vasodilation is mediated via a different VIP receptor or by paracrine VPAC2 stimulation. Also, the demonstration of VPAC2 mRNA in mononuclear inflammatory cells is a novel finding with significant impact on the role of VIP in cutaneous immunomodulation, as a multitude of functional studies have suggested an important role of the peptide in the regulation of the immune system (Bellinger et al., 1996Bellinger D.L. Lorton D. Brouxhon S. Felten S. Felten D.L. The significance of vasoactive intestinal polypeptide (VIP) in immunomodulation.Adv Neuroimmunol. 1996; 6: 5-27Abstract Full Text PDF PubMed Scopus (110) Google Scholar). In conclusion, this study demonstrates an abundant expression of VPAC2 mRNA in human skin that is associated to VIP immunoreactive nerve fibers and suggests a major participation of the VPAC2 receptor in cutaneous VIP-signaling. We thank E. J Goetzl (San Francisco) for generously providing the plasmids. This study was supported by the DFG (SFB 549, C1).