Extracellularly Extruded Syntaxin-4 Is a Potent Cornification Regulator of Epidermal Keratinocytes
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In the skin epidermis, keratinocytes undergo anchorage-dependent cornification, which gives rise to stratified multilayers, each with a distinct differentiation feature. The active formation of the cornified cell envelope (CCE), an important element in the skin barrier, occurs in keratinocytes of the upper epidermal layers and impacts their terminal differentiation. In the present study, we identified the extracellularly extruded syntaxin-4 as a potent differentiation regulator of epidermal keratinocytes. We found that differentiation stimuli led to the acceleration of syntaxin-4 exposure at the keratinocyte cell surface and that the artificial control of extracellular syntaxin-4, either by the forced expression of several syntaxin-4 mutants with structural alterations at the putative functional core site (AIEPQK), or by using antagonistic circular peptides containing this core sequence, dramatically influenced the CCE formation, with spatial misexpression of TGase1 and involucrin. We also found that the topical application of a peptide that exerted the most prominent antagonistic activity for syntaxin-4, named ST4n1, evidently prevented the formation of the hyperplastic and hyperkeratotic epidermis generated by physical irritation in HR-1 mice skin. Collectively, these results demonstrate that extracellularly extruded syntaxin-4 is a potent regulator of CCE differentiation, and that ST4n1 has potential as a clinically applicable reagent for keratotic skin lesions.
The stratified epidermis is constructed through the outward proliferation of keratinocytes and their successive cornification. Epidermal homeostasis is maintained under the strict control of a position-dependent progression of cornification/differentiation and a certain type of apoptosis (anoikis), which leads to dramatic cell-shape changes along with the ultimate exfoliation of the fully cornified scurf at the outermost skin surface (1, 2, 3). Perturbation of the proper cornification program results in a disruption of epidermal homeostasis, causing the onset of skin diseases such as dyskeratosis, atopic dermatitis and psoriasis (2,4, 5, 6). The cornified cell envelope (CCE) is an important element of the skin barrier and a major cornification/differentiation indicator in the middle and upper epidermal layers which is formed as a result of the exclusion of the plasma membrane by lamellar bodies containing abundant ceramide, fatty acids and cholesterol (7). CCE formation is preceded by a steep upregulation and accumulation of CCE structural proteins, such as involucrin, loricrin and envoplakin in the proximal membrane region (7,8). These CCE components and certain keratins are then cross-linked by transglutaminases (TGases) so as to reinforce the CCE structure (7,9,10). Given that epidermal keratinocytes lose the intact plasma membrane structure in accord with this cornification process, the cytoplasmic components anchored to the plasma membrane should be included as candidate elements for the differentiation control in the adjacent cells.
Previously, we showed that the keratinocyte cell line HaCaT extruded a small subpopulation of syntaxin-4 on the cell surface and its forcible expression accelerated CCE formation (11). In the same study, we showed that the circular peptide ST4n1 antagonized the effect of exogenous extracellular syntaxin-4. Syntaxin-4 belongs to the t-SNARE protein family, which mediates the fusion of intracellular vesicles with cell membranes and is abundantly expressed in the epithelial compartment of various tissues including the skin (11, 12, 13). Extracellular localization of syntaxin-4 is similar to that found for the related protein, epimorphin (known also as syntaxin-2), which is produced mainly in the stromal compartment of tissues (14, 15, 16, 17) and which can translocate across the membrane in response to external stimuli to exert its latent signaling functions (18, 19, 20, 21). Similarly, syntaxin-3 effluent from dying keratinocytes was recently found to mediate its extracellular functions (22). Although syntaxin-4 and epimorphin share secondary and tertiary structures (13,23,24), the effects of these two molecules on CCE formation are apparently the opposite in HaCaT keratinocytes: syntaxin-4 induces, while epimorphin inhibits CCE formation (11). However, the elucidation of their biological relevance and the molecular insights still remain unknown.
In the present study, an effort was made to clarify the extracellular role of extruded syntaxin-4 on epidermal differentiation using both normal human keratinocytes and dyskeratotic mice models. Based on the results, insight was obtained into the biological relevance of extracellularly extruded syntaxin-4 and the possible clinical applications of its antagonistic peptide ST4n1.
Materials and Methods
Normal human epidermal keratinocytes (NHEK) were maintained in Keratinocyte Basal medium2 (KGM2) supplemented with SingleQuots (Lonza, Auckland, New Zealand), as per the manufacturer’s protocol. To induce keratinocyte differentiation/cornification, the cells were incubated in KGM2 added with CaCl2 (1 mmol/L) and JNK inhibitor SP600125 (1 µmol/L) for two days, or in KGM2 with the calcium ionophore A23187 (20 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) for 5 h, as described previously (25,26). Functionally and phenotypically normal HaCaT keratinocytes (a gift from M Manabe of Akita University) and 3T3-derived PT67 packaging cells (Clontech, Mountain View, CA, USA), as well as their derivatives, were maintained in DMEM/HamF12 medium (Wako Chemicals, Osaka, Japan) supplemented with 10% FCS along with penicillin and streptomycin (DH10). The human fetal lung fibroblast cell line MRC-5 was maintained in MEM alpha medium (Wako Chemicals) supplemented with 10% FCS. In some wells of NHEK cells, 50 µg/mL of soluble recombinant syntaxin-4 or GFP (11) (recombinant syntaxin-4 is also commercially available from R&D Systems [Minneapolis, MN, USA]) was added to the cells and cultured for 3 d (for the induction of CCE formation), 1 wk (for quantitative real-time PCR analyses) or 2 wks (for the Western blot analyses of TGase1 and involucrin).
Assessment for Cornified Cell Envelope (CCE) Formation Activity
To induce CCE formation in the cultured keratinocytes, NHEK or HaCaT cells suspended in serum-free medium (200,000 cells/mL) were treated with the calcium ionophore A23187 (20 µg/mL) for 5 h at 37°C, washed with PBS, resuspended in PBS containing 2% SDS and 20 mmol/L DTT and boiled for 10 min as reported previously (11,27,28). The number of the remaining hard-shelled cells due to the abundant CCE was counted and the relative value compared with the control was defined as the CCE formation index.
Western blotting, immunohistochemistry and immunocytochemistry were performed according to standard protocols. The primary antibodies include those against TGase1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, for Western blotting, and Biomedical Technologies, Heysham, UK, for immunochemistry), involucrin (Santa Cruz Biotechnology), lamin A/C (Cell Signaling Technology, Tokyo, Japan, and Genetex, Irvine, CA, USA), β-actin (Sigma-Aldrich) and syntaxin-4 (11). Western blotting was carried out with appropriate HRP-labeled second antibodies (GE Healthcare, Pittsburgh, PA, USA) and ECL reagent (Invitrogen [Thermo Fisher Scientific Inc., Waltham, MA, USA]). To selectively detect the cell surface subpopulation of syntaxin-4, a medium of nonpermeabilized living keratinocytes on a chamber slide was added with affinity purified anti-syntaxin-4 antibodies (1/100) (11). After incubation for 1 h, the cells were washed twice with Tris-buffered saline (TBS), fixed with −20°C methanol for 10 min and treated with the secondary second antibodies. As the control, a mouse antibody against lamin A/C was also added to the medium. For immunocytochemistry and immunohistochemistry, the Cy3- or FITC-labeled second antibodies (GE Healthcare) were used with the nuclei counterstained with DAPI (Sigma-Aldrich). The cells were analyzed using the A1 confocal microscope system (Nikon, Tokyo, Japan) or the AXIOSHOP fluorescence microscope (Zeiss Japan, Osaka, Japan) with the VB-7010 CCD camera (Keyence, Osaka, Japan).
Plasmids and Transfection
Expression plasmids for the syntaxin-4 mutants StxΔ4, StxG4 and Stx(2)4, which harbor structural alternations at the putative functional core domain (amino acid number from 103 to 108 [aa 103–108]), were generated using extracellular syntaxin-4 (11) as a template. The cDNAs for the N-terminal interleukin-2 (IL-2) signal peptide, followed by the N-terminal portion of syntaxin-4 with its C-terminal modifications (aa 1–102 for StxΔ4, aa 1–108 plus 4× glycine for StxG4 and aa 1–102 plus epimorphin’s functional core SIEQSC for Stx(2)4), were generated by PCR using a common forward primer containing an EcoRI restriction site and specific reverse primers. The common forward primer was 5′-AAAGAATTCA TGTACAGGATGCAG-3′ and the specific reverse primers were 5′-TTTTAGCTGC GCCCGGACC-3′ (for STΔ4), 5′-TCCTCCTCCTCCTTTTAGCTGCGCCCGGACCTC-3′ (for StxG4) and 5′-ACAGCTCTGCTCAATAGATTTTAGCTGCGCCCGGACC-3′ (for Stx(2)4). The cDNA for the C-terminal portion of syntaxin-4 (aa 109–298) was prepared separately with an EcoRI restriction site at the C-terminus by PCR with the primer pair 5′-GCCATAGAGCCCCAGAAG-3′ and 5′-TTTGAATTCTTATCCAACGGTTAT-3′. The cDNAs for the N- and C-terminal portions were blunt ended, phosphorylated at their 5′ ends and ligated using a BRL kit (Takara, Kusatsu, Japan). The full-length cDNA for each syntaxin-4 derivative was then generated by PCR using the primer pair 5′-AAAGAATTCATGTACAGGATGCAG-3′ and 5′-TTTGAATTCTTATCCAACGGTTAT-3′, treated with EcoRI and cloned into the retroviral expression vector pQCXIN (Invitrogen [Thermo Fisher Scientific]). PT67 packaging cells were transfected with each plasmid using lipofectamine2000 (Invitrogen [Thermo Fisher Scientific]) and subsequently selected for G418 resistance, as described previously (28). To obtain HaCaT cells that stably express each syntaxin-4 derivative, cells were treated with retroviral particles in the supernatant of the PT67 transfectants and grown in the presence of G418 (500 µg/mL).
Quantitative Real-Time PCR (qRT-PCR)
The total RNA was extracted from NHEK cells cultured with r-GFP or r-Stx4 (50 µg/mL) for 1 wk using an RNeasy mini kit (Qiagen, Tokyo, Japan) and was reverse transcribed with ReverTra Ace (TOYOBO, Osaka, Japan). qRT-PCR was performed using FastStart Essential DNA Green Master on LightCycler Nano system (Roche, Basel, Switzerland) according to the manufacturer’s protocol. The primer pairs used in this study were 5′-CCAACCGCGAGAAGATGA-3′ and 5′-CCAGAGGCGTACAGGGATAG-3′ (for β-actin), 5′-ATCAATCTCGGTTGGATTCG-3′ and 5′-TCCGCTTGTTGATTT CATCC-3′ (for keratin1), 5′-CAGCAGCTGGAGTTTGCTAGA-3′ and 5′-GTCCTTCGGATGTCCTCACT-3′ (for envoplakin), and 5′-CAGACAAGATGTCTTATCAGAAAAAGC-3′ and 5′-GAGGTCTTCACGCAGTCCA-3′ (for loricrin). The expression of the mRNA was normalized to that of β-actin and the relative amount to that from GFP-treated cells was measured for each category. The qRT-PCR analyses were performed three times.
3D Skin-Equivalent Model
The three dimensional (3D) skin-equivalent model was prepared as described previously (28) with a slight modification. In brief, type I collagen solution (Type I-AC, Nitta gelatin) containing MRC-5 cells (68,000 cells) was put into a cell-culture insert of 24 well culture plate (Becton, Dickinson and Company [BD], Franklin Lakes, NJ, USA). After incubation at 37°C for 1 h, the upper surface of the resultant collagen gel was coated with 50 ng/mL fibronectin (BD), then seeded with HaCaT cells suspended in DH10 medium (10,000 cells/well). This prepared culture assembly was set in a well of 24-well plate containing DH10 medium and incubated for 1 d. The culture medium was then changed to DH10 medium containing hydrocortisone (0.4 µg/mL) (Sigma-Aldrich), gentamycin (100 µg/mL) (Gibco [Thermo Fisher Scientific]), insulin (5 µg/mL) (Sigma-Aldrich) and ascorbic acid (50 µg/mL) (Sigma-Aldrich). After 3 d, the medium in the upper insert was completely removed so that the apical surface of HaCaT cells was exposed directly to the air. In this culture system, HaCaT cells grew upwardly with successive differentiation within 2 wks, which gave rise to the stratified epidermal tissue on the dermis-like collagen gel.
The circular peptides for the potential antagonists of extracellular syntaxin-4 were generated by the KNC Laboratories (Kobe, Japan). The putative functional core of syntaxin-4 (AIEPQK) was connected with cysteine (ST4n0), cysteine-glycine (ST4n1) or GABA-cysteine (ST4gaba) at the N-terminus and with cysteine at the C-terminus, followed by the introduction of a disulfide bridge between the N- and C-termini. The purity of all of the peptides was more than 97%, as judged by reverse-phase chromatography, and each peptide was added to the culture at a concentration of 1 µg/mL.
Model Mice and the Topical Administration of ST4n1
To prepare the physical irritation-triggered hyperplastic skin, the outer epidermal layers in the dorsal skin of HR-1 female mice (7-wk-old, Japan SLC, Hamamatsu, Japan) were removed by repeated tape stripping (15 times), as described previously (29,30), and the injured skin regenerated a complete, but hyperplastic, epidermis in 5 d. To test the effect of ST4n1, 100 µL of the peptide (10 µg/mL in 50% ethanol) or placebo (50% ethanol) was applied daily onto five or four mice, respectively, for 5 d. After measurement of the moisture of the skin surface with a skin moisture checker (MY-808S, Scalar Corp., Chigasaki, Japan), all the mice were euthanized and the transverse sections of the skin stained with hematoxylin to measure the thickness of the total epidermis as well as the denucleated horny layer. The transverse skin sections were stained with hematoxylin or DAPI. To prepare the dry skin model, six male HR-1 mice (4 wks old) were fed a low-magnesium diet (Hoshino Laboratory Animals, Bando, Japan) as described previously (31). All of the experimental procedures using mice were approved by the Animal Care Committee of Kwansei Gakuin University.
Results are expressed as the mean ± standard deviation (s.d.) of three independent experiments. Data were analyzed using the t test, and p values <0.05 were considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Differentiation-Triggered Extrusion of Syntaxin-4 from NHEK Cells
Effect of Extracellular Syntaxin-4 on CCE Formation of NHEK Cells
Conformation of the Putative Functional Core is Important for CCE Formation Activity
Circular Peptides Generated from the Functional Core of Syntaxin-4 as Potential Antagonists of Extracellular Syntaxin-4
Altered Localization of TGase1 in the Stratified Keratinocytes Induced by Forcible Expression of Extracellular Syntaxin-4 is Reverted by ST4n1
ST4n1 Remedies Hyperkeratosis in the Epidermis of HR-1 Mice
While the role of cytoplasmic syntaxin-4 in vesicular fusion has been investigated extensively, here, we studied its cell surface expression pattern and uncovered a critical role in epidermal differentiation. As in the case of other plasmalemmal syntaxins, epimorphin translocates across the plasma membrane via a nonclassical secretion pathway that is utilized by other leaderless proteins, such as FGF or IL1β (20). We also detected extracellularly effluent syntaxin-3 from keratinocytes undergoing necrotic/apoptotic cell death (22). Although these syntaxins may execute distinctive functions after being exposed extracellularly, they have been commonly shown to elicit survival activity from adjacent cells (11,12,22,34). This function may account for the thickened lower epidermal layers in organ culture and the mice model, the cells of which otherwise progress with an active anoikis program, which may be often driven by the extracellular expression of syntaxin-4. In the later differentiation stages, the keratinocyte cell membrane becomes gradually replaced by a lining of CCE (7), which may provide a means for the active extrusion of membrane-tethered cytoplasmic syntaxin-4. In accord with this, the induction of CCE-formation led to an increased exposure of syntaxin-4 at the cell surface, which in turn appears to support CCE formation in adjacent cells, thereby regulating epidermal cornification.
In the keratinocyte model with HaCaT cells, expression of syntaxin-4 mutants with structural alterations at their functional core site showed loss of their original activity. However, one lacking the functional core (StxΔ4) was selectively downregulated, whereas another possessing with the epimorphin functional core (Stx(2)4) dramatically perturbed the localization of TGase1, both of which induced the severe defect in CCE formation as compared with mock control. The fact that extracellular epimorphin abolished CCE formation (27,35) may account only for the latter case: the functional core of epimorphin may confer the characteristic activity of epimorphin upon syntaxin-4. By contrast, the mechanism for impairment of CCE formation by the overexpression of StxΔ4 is not as clear, except for a dominant-negative effect on syntaxin-4. While it is possible that StxΔ4 associates with endogenous syntaxin-4 via the coiled-coil motifs so as to hinder its activity, or with epimorphin to potentiate its inhibitory effect on CCE formation, further investigation is obviously needed to clarify this issue.
ST4n1, but not ST4 gaba, antagonizes the function of endogenous syntaxin-4, confirming the importance of three dimensional structure of the functional core site. The importance of the corresponding site has been revealed equiponderant also in epimorphin, albeit its effect in CCE formation may be completely opposite (11). Consistent with their complementary role, ST4n1 also exerts the opposite effect as EPn1, the antagonistic peptide generated from the functional core of epimorphin (27). Then how are these proteins from the same family, as well as their antagonistic peptides, able to have such different effects? ST4n1 and EPn1 are comprised of different numbers of distinct peptide motifs, suggesting the existence of a specific receptor for the extracellular forms of syntaxin-4 and epimorphin, each of which propagates a specific signaling pathway. On the other hand, antibodies against integrins blocked the keratinocyte adherence to both epimorphin (18, 19, 20) and syntaxin-4 (data not shown). Thus, it is conceivable that keratinocytes possess receptor complexes for these syntaxins, in which integrins mediate recognition of both syntaxins, while as-yet-unidentified molecules elicit distinct cellular responses.
This study shows that extracellularly extruded syntaxin-4 plays a causal role in CCE formation in keratinocytes and that the syntaxin-4 antagonist ST4n1 may have utility for certain keratotic skin lesions. Together with the previously reported finding that another family member, epimorphin, elicits the opposite cellular response in CCE formation, epidermal differentiation may be critically controlled by extracellularly extruded populations of syntaxin family proteins.
The authors declare they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We are grateful to M Manabe for the HaCaT keratinocyte. We thank DC Radisky and all members of the laboratory for helpful discussions. Part of this work was supported by Grant in Aid for Scientific Research (KAKENHI 24590365).
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