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The Serine Protease Activity of Corin Is Required for Normal Pigment Type Switching

2018; Elsevier BV; Volume: 139; Issue: 1 Linguagem: Inglês

10.1016/j.jid.2018.07.024

1523-1747

Efrat Avigad Laron, Emil Aamar, David Enshell‐Seijffers,

Biochemical Analysis and Sensing Techniques

The production and deposition of pigment in mouse skin are confined to the hair follicle and hair shaft, respectively. Hair pigmentation in mice involves the deposition of two types of pigment, black (eumelanin) and yellow (pheomelanin), in a very specific pattern (Barsh et al., 2000Barsh G. Gunn T. He L. Schlossman S. Duke-Cohan J. Biochemical and genetic studies of pigment-type switching.Pigment Cell Res. 2000; 13: 48-53Crossref PubMed Scopus (65) Google Scholar). Pigment, either black or yellow, is synthesized during the growth phase (anagen) of the hair cycle by melanocytes that reside in the hair bulb surrounding the upper half of the dermal papilla (DP), a specialized mesenchymal compartment that plays important roles in regulating different aspects of hair follicle biology. Keratinocytes in the hair bulb that undergo differentiation to form the building blocks of the hair shaft take up pigment from nearby melanocytes, leading to the formation of pigmented hair. Activity of Mc1r receptor in melanocytes results in black pigment production (Jackson et al., 2007Jackson I.J. Budd P.S. Keighren M. McKie L. Humanized MC1R transgenic mice reveal human specific receptor function.Hum Mol Genet. 2007; 16: 2341-2348Crossref PubMed Scopus (34) Google Scholar, Slominski et al., 2005Slominski A. Plonka P.M. Pisarchik A. Smart J.L. Tolle V. Wortsman J. et al.Preservation of eumelanin hair pigmentation in proopiomelanocortin-deficient mice on a nonagouti (a/a) genetic background.Endocrinology. 2005; 146: 1245-1253Crossref PubMed Scopus (104) Google Scholar, Smart and Low, 2003Smart J.L. Low M.J. Lack of proopiomelanocortin peptides results in obesity and defective adrenal function but normal melanocyte pigmentation in the murine C57BL/6 genetic background.Ann NY Acad Sci. 2003; 994: 202-210Crossref PubMed Scopus (54) Google Scholar). Binding of Agouti to Mc1r reduces Mc1r signaling and switches the production from black pigment to yellow (Ollmann et al., 1998Ollmann M.M. Lamoreux M.L. Wilson B.D. Barsh G.S. Interaction of agouti protein with the melanocortin 1 receptor in vitro and in vivo.Genes Dev. 1998; 12: 316-330Crossref PubMed Scopus (189) Google Scholar). During early anagen, a sharp peak of Agouti expression in DP cells is observed (Millar et al., 1995Millar S.E. Miller M.W. Stevens M.E. Barsh G.S. Expression and transgenic studies of the mouse agouti gene provide insight into the mechanisms by which mammalian coat color patterns are generated.Development. 1995; 121: 3223-3232Crossref PubMed Google Scholar). This peak generates a short and provisional period in which Mc1r activity is suppressed by Agouti and temporarily switches the melanocytes to produce pheomelanin. This creates a subapical yellow band in an otherwise black hair, resulting in an overall appearance of a mottled brown hair coat. The interaction between Mc1r and Agouti is modified by Corin (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). Corin encodes a type II transmembrane serine protease that is expressed specifically in the DP and adjusts Agouti inhibition by narrowing the window of effective Agouti activity. In the absence of Corin, Agouti activity is prolonged and the yellow band is extended, leading to lighter coat color. Because Corin is a type II transmembrane serine protease and its single-pass transmembrane domain resides in close proximity to the N-terminus, most of Corin is extracellular (Figure 1a). In addition to regulating pigment type switching, Corin plays important role in blood pressure regulation. The protease activity of Corin in the heart cleaves the prohormone Nppa to its active form and thus activates the natriuretic peptide pathway to control blood tension (Chan et al., 2005Chan J.C. Knudson O. Wu F. Morser J. Dole W.P. Wu Q. Hypertension in mice lacking the proatrial natriuretic peptide convertase corin.Proc Natl Acad Sci USA. 2005; 102: 785-790Crossref PubMed Scopus (207) Google Scholar, Yan et al., 2000Yan W. Wu F. Morser J. Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.Proc Natl Acad Sci USA. 2000; 97: 8525-8529Crossref PubMed Scopus (381) Google Scholar). In the uterus, Corin activates Nppa to regulate blood pressure during pregnancy by augmenting trophoblast invasion and remodeling spiral arteries, thus preventing preeclampsia (Cui et al., 2012Cui Y. Wang W. Dong N. Lou J. Srinivasan D.K. Cheng W. et al.Role of corin in trophoblast invasion and uterine spiral artery remodelling in pregnancy.Nature. 2012; 484: 246-250Crossref PubMed Scopus (220) Google Scholar). In contrast, little is known about the molecular mechanism by which Corin acts to inhibit Agouti activity and regulate pigment type switching. It was speculated that the protease activity of Corin mediates the Corin role in pigment type switching, but direct evidence for such a mechanism is lacking (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). In contrast to Corin’s role in blood pressure regulation, Corin may regulate Agouti activity by a different mode of action that is unrelated to its serine protease activity. In such model, Corin may act as a receptor that transduces signaling into DP cells to modify Agouti activity or as extracellular inhibitor that sequesters the activity of Agouti. To explore whether the serine protease activity of Corin is required in the regulation of pigment type switching, the serine protease activity of Corin was abolished while preserving its complex structure. Using a gene targeting approach, a missense mutation that substitutes the serine (S) residue of the catalytic triad to alanine (A) was introduced (Figure 1b and c). This alteration has been previously shown in vitro to diminish the catalytic activity of Corin without affecting its surface localization and stability (Yan et al., 2000Yan W. Wu F. Morser J. Wu Q. Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme.Proc Natl Acad Sci USA. 2000; 97: 8525-8529Crossref PubMed Scopus (381) Google Scholar). Furthermore, this substitution was also designed to introduce the SgrA I restriction site to allow routine genotyping. A targeting construct was generated to include the S/A substitution and transfected into embryonic stem cells. Chimeric mice were derived from correctly targeted embryonic stem cell clones and used to establish a mouse line that harbors the S/A substitution. Wild-type, heterozygous, and homozygous mice for the missense mutation were designated CorinS/S, CorinS/A, and CorinA/A, respectively. Primers that flank the SgrA I site were designed to PCR-amplify a fragment of 390 base pairs (Figure 1b). In the case of the CorinA allele, digestion of the PCR product resulted in two fragments of 118 base pairs and 272 base pairs. This way, the CorinS and CorinA alleles were easily distinguished in agarose gel electrophoresis after digestion with SgrA I (Figure 1d). Similar to mice with a null allele of Corin (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar), mice homozygous for the CorinA allele were viable, fertile, and recovered at expected Mendelian frequencies. Furthermore, as expected, follicle morphology and the hair cycle appeared normal. Homozygous mutants were also assessed in vivo for the presence of the Corin mutant in the DP of the hair follicle in skin sections using immunostaining with anti-Corin antibodies (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar). Because the missense mutation is not expected to affect Corin levels and structure, these antibodies detected apparently normal levels of Corin in the DP of homozygous CorinA/A mutant mice (Figure 2a and b). Phenotypic analysis of coat color on normal Agouti background showed that mice homozygous for the CorinA allele display a distinctively lighter coat color (Figure 2c–e). Similar to the null phenotype (Enshell-Seijffers et al., 2008Enshell-Seijffers D. Lindon C. Morgan B.A. The serine protease corin is a novel modifier of the agouti pathway.Development. 2008; 135: 217-225Crossref PubMed Scopus (92) Google Scholar), this coat color phenotype is most pronounced in juveniles (Figure 2c). Hair shafts were plucked from the back skin of wild-type and mutant mice at the end of the first hair cycle and analyzed microscopically (Figure 2e). Mice homozygous for the CorinA allele exhibit extended subapical yellow band. This clearly illustrates that the serine protease activity of Corin is required for inhibiting Agouti activity during pigment type switching. A missense mutation in the Corin gene of the golden tiger has been recently identified (Xu et al., 2017Xu X. Dong G.X. Schmidt-Kuntzel A. Zhang X.L. Zhuang Y. Fang R. et al.The genetics of tiger pelage color variations.Cell Res. 2017; 27: 954-957Crossref PubMed Scopus (15) Google Scholar). This mutation results in the substitution of a histidine residue into tyrosine in the sixth LDLR domain of Corin and therefore does not interfere with its protease activity. However, this genetic alteration results in extremely elongated yellow band and, consequently, a light-colored tiger. Although the ability of Corin to proteolytically cleave Agouti has not been directly tested in that study and therefore precluded the assessment of Agouti as a direct substrate of Corin, this analysis may suggest that the underlying mechanism by which Corin interacts with Agouti is similar to the way Corin regulates proANP in the heart (Knappe et al., 2004Knappe S. Wu F. Madlansacay M.R. Wu Q. Identification of domain structures in the propeptide of corin essential for the processing of proatrial natriuretic peptide.J Biol Chem. 2004; 279: 34464-34471Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Initially, a direct protein-protein interaction between the LDLR domains of Corin and Agouti recruits Agouti to the vicinity of the protease domain, and subsequently, the serine protease activity of Corin inactivates Agouti by proteolytic cleavage. Unfortunately, the low and variable level of Agouti protein in the skin and the limited sensitivity of available antisera prevent the in vivo assessment of direct interaction between Agouti and Corin and the proteolytic consequences of this interaction on Agouti. Only future development of novel tools will allow us to molecularly address the mechanism by which Corin and Agouti interact. Mice were housed according to Federation of Laboratory Animal Science Associations guidelines. All mice were bred and maintained in a temperature-controlled room, on a 12-hour light-dark cycle, and with food and water available ad libitum. All experimental protocols were approved by the Animal Care and Use Committee of Bar Ilan University. The authors state no conflict of interest. This research was funded by the United States-Israel Binational Science Foundation (BSF: 2013375) and the Israel Science Foundation (ISF: 1304/14). We thank Hofesh Haruach for technical support. Download .pdf (.07 MB) Help with pdf files Supplementary Material