Exp Dermatol 2001: 10: 193–203 Copyright C Munksgaard 2001 Printed in Denmark Á All rights reserved EXPERIMENTAL DERMATOLOGY ISSN 0906-6705 Proprotein convertase expression and localization in epidermis: evidence for multiple roles and substrates Pearton DJ, Nirunsuksiri W, Rehemtulla A, Lewis SP, Presland RB, Dale David J. Pearton1, BA. Proprotein convertase expression and localization in epidermis: evi- Wilas Nirunsuksiri1,3, dence for multiple roles and substrates. Alnawaz Rehemtulla2, C Exp Dermatol 2001: 10: 193–203. Munksgaard, 2001 S. Patrick Lewis1, Richard B. Presland1 and Abstract: Specific proteolysis plays an important role in the terminal dif- 1 ferentiation of keratinocytes in the epidermis and several types of proteases Beverly A. Dale have been implicated in this process. The proprotein convertases (PCs) 1Departments of Oral Biology, Periodontics, are a family of Ca2¹-dependent serine proteases involved in processing and Biochemistry, Medicine/Dermatology, University 2 activation of several types of substrates. In this study we examined the of Washington, Seattle, WA 98195; Department of Radiation Oncology, University of Michigan, expression and some potential substrates of PCs in epidermis. Four PCs Ann Arbor, MI; 3Present address Dow are expressed in epidermis: furin, PACE4, PC5/6 and PC7/8. Furin is de- AgroSciences LLC, Indianapolis, IN tected in two forms, either with or without the transmembrane domain, suggesting occurrence of post-translational cleavage to produce a soluble enzyme. In addition the furin active site has differential accessibility in the granular layer of the epidermis relative to the basal layer, whereas antibodies to the transmembrane domain stain both layers. These find- ings suggest that furin has access to different types of substrates in granu- Key words: epidermal differentiation – lar cells as opposed to basal cells. PC7/8, in contrast, is detected throughout keratinocytes – furin – PACE4 – PC5/PC6 – the epidermis with antibodies to both the transmembrane and active site PC7/PC8 – profilaggrin – Notch-1 and no soluble form observed. A peptide PC inhibitor (dec-RVKR-CMK) Dr Beverly A. Dale, Dept of Oral Biology, inhibits cleavage of Notch-1, a receptor important in cell fate determi- University of Washington, Box 357132, Seattle, nation that is found throughout the epidermis. Profilaggrin, found in WA 98195 7132 the granular layer, is specifically cleaved by furin and PACE4 in vitro at Tel.: 206 543 4393 a site between the amino terminus and the first filaggrin repeat. This Fax: 206 685 3162 work suggests that the PCs play multiple roles during epidermal differen- e-mail: bdale/u.washington.edu tiation. Accepted for publication 12 January 2001 tified that play a role in epidermal differentiation. Introduction These include calpain I (1), cathepsins C (2, 3), B, The sequential differentiation of keratinocytes in H and L (4), profilaggrin endopeptidase (PEP1) the epidermis is a complex, highly regulated series (5), the stratum corneum chymotryptic-like pro- of events that results in the formation of the anu- tease (SCCP) and the stratum corneum thiol pro- cleate cornified cells of the stratum corneum from tease (SCTP) (6). The importance of proteases in the relatively undifferentiated cells of the basal the epidermis is highlighted by the finding that null layer. This differentiation process is calcium de- mutations of cathepsin C result in palmoplantar pendent and requires extensive and tightly con- keratoderma disorders, specifically Haim–Munk trolled changes in both protein expression and en- and Papillon–Lefevre syndromes (2, 7, 8). zymatic activities resulting in characteristic A protease family that has been implicated in morphological changes of the cells. Proteolytic processing and differentiation in a number of processing is an important mechanism for many of tissues is the Proprotein Convertase (PC) family. these events, including signaling, proprotein acti- This is a family of Ca2¹-dependent serine pro- vation, structural remodeling and desquamation. teases related to bacterial subtilisin (for reviews see A number of different proteases have been iden- 9–12). To date 7 family members have been iden- 193 Pearton et al. tified; furin (also known as PACE), PC2, PC1/3, ¶60–70% of confluence, at 100% confluence or PACE4, PC4, PC5/6 and PC7/8/LPC. Of these fur- several days post-confluence. in, PACE4, PC5/6 and PC7/8 are widely expressed whereas PC2 and PC1/3 are limited to neuro-endo- Detection of proprotein convertase expression in crine tissues and PC4 is restricted to the testis, sug- skin using degenerate primers gesting both generalized and tissue-specific pat- terns of expression and function. The PC enzymes The sequences of the known human PC members recognize basic motifs, cleaving after paired basic (furin, PACE4, PC1, PC2, PC5/6, PC7/8) were ob- residues (PC2 and PC1/3) or after a canonical tained from Genbank using the ENTREZ server. RX(R/K)R motif (furin and PACE4); in some sub- The sequences were multiply aligned using Clus- strates a P6 Arg can substitute for the Arg at the talW (19, 20) and a highly conserved region of the P4 position (13). Furin has been shown to process catalytic domain was identified to which degener- a wide variety of substrates including receptors, ate primers were designed. The sense primer (5¿- growth factors, hormones, plasma proteins, matrix CA(C/T) GG(C/G) AC(A/T/G/C) (A/C) G(A/T/G/ metalloproteinases and extracellular matrix com- C) TGTGC (A/T/G) GG-3¿) was based on the se- ponents. In addition PC family members are also quence R194HGTRCAG201 in furin which contains involved in the processing of exogenous proteins the active site arginine (R194). This sequence is such as some viral envelope glycoproteins and bac- 100% conserved across the known human mem- terial endotoxins (12). bers of the PC family and highly conserved in Several proteins relevant to keratinocyte devel- other species. The antisense primer (5¿-GG (T/G) opment have been shown to be substrates for PC CCC CAG CT (T/G) (C/G) (A/C) (A/G) CTG TA- processing or contain potential PC cleavage sites. 3¿) was based on the sequence I249YSASWG255 These include receptors such as Notch-1 (14, 15), which is also highly conserved across the family, matrix components such as collagen XVII (BP180) although in PC7/8 the alanine (A252) is replaced (16), matrix metalloproteinases, desmosomal com- by a cysteine. RT-PCR using these primers would ponents such as desmoplakin-1 and desmoglein, amplify a 187 bp segment from each PC that could integrins including integrins a3 and a6 (17) and be distinguished on the basis of its nucleotide se- structural proteins such as profilaggrin. quence. Although PCs have diverse substrates and play Total RNA was isolated from 5-day post-con- a role in differentiation in some systems there has fluent cultured human keratinocytes using the Tri- been no systematic study of PC expression in epi- zol reagent (Life Technologies, Gaithersburg, MA, dermis (2). We therefore investigated the express- USA) and the manufacturer’s protocol. After ion and distribution of the members of the PC DNase I treatment the total RNA was reverse family in epidermis and examined the processing transcribed using the Superscript II RT system of several potential substrates. (Life Technologies) and the resulting cDNA was In this study, 4 members of the PC family (furin, PCR amplified (40 cycles at 95æC for 30 s; 60æC PACE4, PC5/6 and PC7/8) were detected in epider- for 1 min; 72æC for 1 min) using Taq plus Taq mis. Furin and PC7/8 were detected in distinct, al- extender (Promega, Madison, WI, USA). The re- though overlapping, patterns within the epidermis. sulting 187 bp product was gel purified and TA In addition the active site of furin showed differen- cloned into the pCR3.1 vector (Invitrogen, tial accessibility during differentiation. Inhibition Carlsbad, CA, USA). of the PC family in culture inhibited the processing of Notch-1 and in vitro results characterized a fur- Northern analysis in/PACE4 processing site in the amino terminus of profilaggrin. The specific localization pattern, cal- Total RNA was isolated from human foreskin cium dependence, and identification of possible keratinocytes according to the method of Chom- substrates are consistent with a role for PCs in czynski & Sacchi (21), fractionated on glyoxal gels, multiple events during epidermal differentiation. transferred to Genescreen Plus (Du Pont NEN, Boston, MA, USA) by capillary blotting according to manufacturer’s recommendation, and hybrid- Methods ized with radiolabeled cDNA probes specific for Keratinocyte culture the coding region of different members of the PC family (22, 23). The blots were subsequently hy- Human foreskin keratinocytes were cultured using bridized with a GAPDH probe and the ratio be- the methods described by Fleckman et al. (18), a tween furin and GAPDH determined by Phos- modification of the Rheinwald–Green system. phorimager (Molecular Dynamics, Sunnydale, Cells were harvested for RNA or protein at either CA, USA). 194 PC expression and localization in epidermis RT-PCR analysis of PC5/6 and PC7/8 0.1% trypsin for a variable length of time (usually 3 min). After preblocking the sections with serum Oligoprimers specific for PC5/6 and PC7/8/LPC specific for the secondary antibodies used, the sec- were made according to published sequences (Gen- tions were incubated with the primary antibodies bank acc. NM–004716). For PC5/6 the sense in TBS containing 1% BSA overnight at 4æC. The primer used comprised nucleotides 962–982 (5¿- primary antibodies were detected with either TCA GCA GCA CTG CAG AAA GC-3¿) and the FITC-conjugated secondary antibodies (Vector, antisense primer, nucleotides 1409–1429 (5¿-TGG Eugene, OR, USA) or biotin-conjugated second- GGT CTT CAA CTC GGC TA-3¿), giving a 447 ary antibodies followed by streptavidin-conjugated bp product. For PC7/8 the sense primer 163–183 Texas Red (Vector).