Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m801844200
ISSN1083-351X
AutoresMargarethe Müller, Kirill Essin, Kerstin Hill, Heike A. Beschmann, Simone Rubant, Christoph M. Schempp, Maik Gollasch, Wolf‐Henning Boehncke, Christian Harteneck, Wernér E.G. Müller, Kristina Leuner,
Tópico(s)Herbal Medicine Research Studies
ResumoThe protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation. The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation. Our skin undergoes constant regulation to provide a protective epithelial barrier, whereby the differentiation of keratinocytes from proliferating cells in the basal layer into platted, dead cells in the stratum corneum plays a major role. Keratinocytes leaving the basal layer not only change structure and shape but also loose their ability to proliferate and enter the terminal differentiation stage by expressing proteins required for the cornification like keratin 1 (K1) 2The abbreviations used are: K1, keratin 1; K10, keratin 10; IVL, involucrin; AD, atopic dermatitis; TRPC, canonical transient receptor potential; TRPV, vanilloid-like transient potential channel; hPK, human primary keratinocytes; YFP, yellow fluorescent protein; DN, dominant negative; RT, reverse transcription; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; RNAi, RNA interference; MES, 4-morpholineethanesulfonic acid. 2The abbreviations used are: K1, keratin 1; K10, keratin 10; IVL, involucrin; AD, atopic dermatitis; TRPC, canonical transient receptor potential; TRPV, vanilloid-like transient potential channel; hPK, human primary keratinocytes; YFP, yellow fluorescent protein; DN, dominant negative; RT, reverse transcription; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; RNAi, RNA interference; MES, 4-morpholineethanesulfonic acid. or keratin 10 (K10) and additional structural proteins that are needed for the cornification such as involucrin (IVL) or transglutaminase (1Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (510) Google Scholar). The precise balance between proliferation and differentiation found in healthy individuals is altered in individuals suffering from various skin diseases like psoriasis or atopic dermatitis (AD). Psoriasis is marked by thickened epidermis because of increased proliferation but disturbed differentiation of keratinocytes (2Lowes M.A. Bowcock A.M. Krueger J.G. Nature. 2007; 445: 866-873Crossref PubMed Scopus (1358) Google Scholar). Accordingly, psoriatic skin is characterized by reduced differentiation markers but elevated epidermal proliferation markers (3Bovenschen H.J. Seyger M.M. Van De Kerkhof P.C. Br. J. Dermatol. 2005; 153: 72-78Crossref PubMed Scopus (88) Google Scholar). Psoriatic plaques are covered by a thick layer of scales caused by aberrant terminal differentiation. The granular layer of the epidermis, in which the terminal differentiation begins, is greatly reduced or absent in psoriatic lesions, which results in a stratum corneum consisting of incompletely differentiated keratinocytes. As for psoriasis, the exact pathomechanisms of AD are not known (4Williams H.C. N. Engl. J. Med. 2005; 352: 2314-2324Crossref PubMed Scopus (421) Google Scholar). In addition to an enhanced immune response, a reduced integrity of the epidermal barrier seems to be relevant. Proksch et al. (5Proksch E. Folster-Holst R. Jensen J.M. J. Dermatol. Sci. 2006; 43: 159-169Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) suggested that a disturbed differentiation of keratinocytes causes a defective skin barrier function in AD, enabling the enhanced penetration of environmental allergens. An impaired expression of cornified envelope proteins like involucrin and keratins like K10, important for permeability barrier formation, could also be detected in lesional skin from patients with AD (6Jensen J.M. Folster-Holst R. Baranowsky A. Schunck M. Winoto-Morbach S. Neumann C. Schutze S. Proksch E. J. Investig. Dermatol. 2004; 122: 1423-1431Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). However, other factors not directly related to keratinocyte function seem to contribute to the altered skin barrier seen in AD patients, e.g. a mutation in the structural protein filaggrin (7Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. Goudie D.R. Sandilands A. Campbell L.E. Smith F.J. O'Regan G.M. Watson R.M. Cecil J.E. Bale S.J. Compton J.G. DiGiovanna J.J. Fleckman P. Lewis-Jones S. Arseculeratne G. Sergeant A. Munro C.S. El Houate B. McElreavey K. Halkjaer L.B. Bisgaard H. Mukhopadhyay S. McLean W.H. Nat. Genet. 2006; 38: 441-446Crossref PubMed Scopus (2208) Google Scholar). Because of the crucial role of keratinocyte differentiation for normal skin function and as relevant pathomechanism in various skin diseases, an exact knowledge of the mechanism relevant for the specific and tight sequence of events leading to keratinocytes proliferation and differentiation is very much needed. On a cellular level, several studies clearly showed that Ca2+ plays a crucial role in the regulation of keratinocyte differentiation especially for the terminal stages like cell stratification and cornification (8Tu C.L. Oda Y. Komuves L. Bikle D.D. Cell Calcium. 2004; 35: 265-273Crossref PubMed Scopus (99) Google Scholar). Induction of differentiation and inhibition of proliferation are tightly regulated by an increase in [Ca2+]i because of both Ca2+ release and Ca2+ influx mechanisms with a still unknown molecular basis. In tissue culture, the differentiation of keratinocytes can be triggered by experimentally increasing [Ca2+]o above 0.1 mm (9Eckert R.L. Crish J.F. Robinson N.A. Physiol. Rev. 1997; 77: 397-424Crossref PubMed Scopus (337) Google Scholar). In a first step, this elevation in [Ca2+]o induces a rise in [Ca2+]i by activating the Ca2+-sensing receptor, a G-protein-coupled receptor (10Tu C.L. Chang W. Bikle D.D. J. Investig. Dermatol. 2007; 127: 1074-1083Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In the next step, stimulation of the Ca2+-sensing receptor activates the phospholipase C pathway generating inositol 1,4,5-triphosphate and diacylglycerol (8Tu C.L. Oda Y. Komuves L. Bikle D.D. Cell Calcium. 2004; 35: 265-273Crossref PubMed Scopus (99) Google Scholar). Both intracellular second messengers elevate intracellular Ca2+ concentration. Inositol 1,4,5-triphosphate as a ligand of inositol 1,4,5-triphosphate receptors induces the release of Ca2+ from the endoplasmic reticulum. Diacylglycerol directly activates members of the canonical transient receptor potential (TRPC) channel family. Based on the sequence homology, activation mechanism, and ability to form heteromeric channel complexes, the proteins of the TRPC group can be divided into the TRPC1, TRPC4, and TRPC5 and the TRPC3, TRPC6, and TRPC7, of which diacylglycerol directly activates only TRPC3, TRPC6, and TRPC7 (11Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1228) Google Scholar). However, the data about the specific TRPC channels relevant for keratinocyte differentiation are controversial. For example Cai et al. (12Cai S.W. Fatherazi S. Presland R.B. Belton C.M. Izutsu K.T. J. Dermatol. Sci. 2005; 40: 21-28Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) detected TRPC1, TRPC5, TRPC6, and TRPC7 in gingival keratinocytes, whereas Beck et al. (13Beck B. Zholos A. Sydorenko V. Roudbaraki M. Lehen'kyi V. Bordat P. Prevarskaya N. Skryma R. J. Investig. Dermatol. 2006; 126: 1982-1993Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) showed the expression of TRPC1, TRPC4, TRPC5, and TRPC7 in HaCaT keratinocytes. Similarly, TRPC1 as well as TRCP4 have been implicated in the Ca2+-sensing receptor triggered elevation of [Ca2+]i (14Cai S. Fatherazi S. Presland R.B. Belton C.M. Roberts F.A. Goodwin P.C. Schubert M.M. Izutsu K.T. Pflúgers Arch. Eur. J. Physiol. 2006; 452: 43-52Crossref PubMed Scopus (60) Google Scholar, 15Fatherazi S. Presland R.B. Belton C.M. Goodwin P. Al Qutub M. Trbic Z. Macdonald G. Schubert M.M. Izutsu K.T. Pflúgers Arch. Eur. J. Physiol. 2007; 453: 879-889Crossref PubMed Scopus (30) Google Scholar). Moreover, following Ca2+-stimulated differentiation of gingival keratinocytes, elevated expression of TRPC1, TRPC5, TRPC6, and TRPC7 has been reported (12Cai S.W. Fatherazi S. Presland R.B. Belton C.M. Izutsu K.T. J. Dermatol. Sci. 2005; 40: 21-28Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The attempts to identify the Ca2+ channels playing the major role for Ca2+-sensing receptor-mediated keratinocyte differentiation have been significantly hampered by the lack of pharmacological tools specifically affecting individual TRPC channel function. However, because we have recently identified hyperforin as a specific and potent TRPC6 activator (16Leuner K. Kazanski V. Múller M. Essin K. Henke B. Gollasch M. Harteneck C. Múller W.E. FASEB J. 2007; 21: 4101-4111Crossref PubMed Scopus (185) Google Scholar, 17Treiber K. Singer A. Henke B. Múller W.E. Br. J. Pharmacol. 2005; 145: 75-83Crossref PubMed Scopus (61) Google Scholar), we were able for the first time to investigate in detail the specific contribution of this channel for Ca2+-mediated keratinocyte differentiation. Our findings not only show that TRPC6 plays a role but also demonstrate that the specific activation of TRPC6 alone is sufficient for nearly full physiological response. TRPC6 activation by hyperforin or similar compounds therefore represents a novel approach to pharmacologically activated keratinocyte differentiation. To elucidate the molecular mechanism for keratinocyte differentiation in culture, we used HaCaT cells as established and characterized cell model and human primary keratinocytes (hPKs) and human skin explants as native systems to validate our data. By this approach, we were able to show that both cell types express functionally active TRPC6 channels in vitro and ex vivo. Furthermore, the use of hyperforin, the recently identified selective activator of TRPC6, enabled us to show that the Ca2+-induced differentiation of keratinocytes is to a large extent mediated by TRPC6 channels. The elucidation of this molecular pathway has several clinical implications. First, the TRPC6 gene is an interesting candidate gene for genetic approaches, and second stimulating TRPC6 channels may be a novel treatment approach in dermatology. Sources and Preparation of Reagents—Hyperforin was a kind gift from Dr. Willmar Schwabe (Karlsruhe, Germany). Fluorescence dyes (SBFI-AM and fura-2-AM) were purchased from Molecular Probes (Eugene, OR). Pluronic F-127, 2-aminophenoxyborate (Tocris, Abvonmouth, UK), and SK&F 96365 (Biotrend, Cologne, Germany) were used from 10 mm stock solution in dimethyl sulfoxide. N-(p-Amylcinnamoyl) anthranilic acid (Calbiochem, San Diego, CA) was used from 50 mm stock solution in dimethyl sulfoxide. GdCl3 and LaCl3 (Sigma-Aldrich) were dissolved in H2O prior to experiments. Cell Culture—The HaCaT human keratinocyte cell line was cultured in keratinocyte-SFM medium (Invitrogen) with 10% heat-inactivated fetal calf serum (Sigma-Aldrich), 50 units/ml penicillin (Sigma-Aldrich), and 50 μg/ml streptomycin (Sigma-Aldrich). Human primary keratinocytes were derived from adult skin and cultured according to the method of Rheinwald and Green (18Rheinwald J.G. Green H. Cell. 1975; 6: 317-330Abstract Full Text PDF PubMed Scopus (409) Google Scholar) in keratinocyte growth medium (Promo Cell, Heidelberg, Germany). HaCaT cells and hPKs were cultured under a 5% CO2 humidified atmosphere at 37 °C. For the experiments, the cells were seeded in 6-well plates for RT-PCR and Western blot and on glass coverslips for histochemistry and Ca2+ imaging. For differentiation studies, the cells were allowed to attach for 24 h after trypsinization, and then 0.1 mm Ca2+-containing keratinocyte-SFM medium was replaced by SFM medium with 2 mm Ca2+ or hyperforin 1 μm. After 48–72 h of incubation in the latter medium, histochemical staining, RT-PCR, and Western blotting of corresponding markers were performed. Split Thickness Skin Organ Culture—6-mm punch biopsies containing epidermis and papillary dermis were obtained from dermatome-separated human skin. The biopsies were floated on SFM in six-well plates in the presence of Ca2+-free medium (negative control), 2 mm Ca2+ (positive control), or 1 μm hyperforin. After 24 h the cultures were terminated, fixed in paraformaldehyde, and embedded in paraffin. 3-μm sections were stained for TRPC6 using the labeled streptavidin biotin method as described (19Schempp C.M. Kiss J. Kirkin V. Averbeck M. Simon-Haarhaus B. Kremer B. Termeer C.C. Sleeman J. Simon J.C. Planta Med. 2005; 71: 999-1004Crossref PubMed Scopus (45) Google Scholar). Five random fields of sections from four independent skin explants were counted for TRPC6-positive keratinocytes at ×400 magnification. The final count/group represents the mean ± S.D. Cell Transfection—HaCaT keratinocytes and hPKs were plated in 6-well plates onto glass coverslips and transiently transfected 24 h later by the addition of a transfection mixture containing 0.5 μg of DNA and 1 μl of FuGENE 6 transfection reagent (Roche Applied Science) in 97 μl of Opti-MEM medium (Invitrogen). The cDNA constructs have been kindly provided by Dr. Michel Schaefer (11Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1228) Google Scholar). Ca2+ imaging was conducted 2 days after transfection. Histochemical staining, RT-PCR, and Western blotting were conducted 2–3 days after transfection. For TRPC knockdown studies with siRNA, HaCaT cells were plated in 6-well plates onto glass coverslips and transiently transfected 24 h later by the addition of transfection mixture containing 100 nm TRPC6 siRNA (Invitrogen) or 25 nm TRPC1, TRPC3, TRPC4, TRPC5, and TRPC7 siRNA (Ambion) and 2.5 μg/ml Lipofectamine 2000 (Invitrogen) in 250 μl of Opti-MEM medium. As a control 100 nm siRNA control sequence with low GC content (Invitrogen) or 25 nm negative RNAi control (Ambion) with their complementary sequences were transfected in the same procedure. Histochemical staining and Western blotting were performed 2–3 days after transfection. RT-PCR—RNA was isolated using TRIzol reagent (Invitrogen), chloroform, and 100% ethanol according to the manufacturer's instructions. The reactions were carried out using 2 μg of mRNA. First strand cDNA was synthesized from 2 μg of total RNA in a 20-μl final volume using a first strand cDNA synthesis kit (Invitrogen). After reverse transcription, amplification was carried out by PCR using Taq DNA polymerase and dNTP set of Invitrogen. A 2-μl aliquot of the reverse transcription solution was used as a template for specific PCR. The PCR primers used to amplify TRPC1, 3, 4, 5, 6, and 7 channels, IVL, TGM I, K1, and K10 cDNAs are specified in Table 1. Commercially available 18 S rRNA primers (Ambion, Huntington, UK) were used as internal loading control, and the predicted 18 S (Classic II) band size was 324 bp. The PCR was conducted under the following conditions: an initial denaturation step at a temperature of 94 °C for 5 min and 30 cycles as follows: 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C, and finally 7 min at 72 °C. PCR products were run on a 1% agarose gel and stained with ethidium bromide. Changes in relative mRNA levels were obtained by relating each PCR product to its internal control. After gel electrophoresis, quantification was archived with Easywin 32 software (Herolab). RT-PCR analysis using TRPC6-specific primer resulted in a fragment of the expected size of 322 bp. The sequence of fragment was sequenced (Seqlab, Göttingen, Germany) and corresponded to the TRPC6 sequence available in GenBank™ under accession number AF080394.TABLE 1Primer and siRNA sequencesNo.NameAccession no.Forward (5′ → 3′)Backward (5′ → 3′)Expected sizebp1K1NM_006112GGACATGGTGGAGGATTACCGTGCTCTTCTGGGCTATATCCTCG3162K10NM_000421GCAAAATCAAGGAGCGGTATGAGAGCTGCACACAGTAGCGACC6853IVLNM_005547TCTAAGATGTCCCAGCAACACATCATGCTGTTCCCAGTGCTGTT2924TGM INM_000359GATCGCATCACCCTTGAGTTACTCCTCATGGTCCACGTACACAAT3045TRPC1Z73903ATGTATACAACCAGCTCTATCTTGAGTCTTTGGTGAGGGAATGATG5256TRPC3U47050CTGCAAATGAGAGCTTTGGCAACTTCCATTCTACATCACTGTC3887TRPC4AF175406ATTCATATACTGCCTTGTGTTGGGTCAGCAATCAGTTGGTAAG3298TRPC5AF054568ACTTCTATTATGAAACCAGAGCGCATGATCGGCAATAAGCTG2899TRPC6AF080394AAGACATCTTCAAGTTCATGGTCTCAGCGTCATCCTCAATTTCC32210TRPC7AJ272034GTCCGAATGCAAGGAAATCTTGGGTTGTATTTGGCACCTC47711TRPC1 siRNA5′-GAUCUGUCAAAAUUCCGAATT-3′5′-UUCGGAAUUUUGACAGAUCTT-3′12TRPC3 siRNA5′-CGUUAUCAGCAGAUAAUGATT-3′5′-UCAUUAUCUGCUGAUAACGTG-3′13TRPC4 siRNA5′-GUAUAGACUAUGAUCUAAATT-3′5′-UUUAGAUCAUAGUCUAUACTA-3′14TRPC5 siRNA5′-CACCCUGAUUCAUCCGAGATT-3′5′-UCUCGGAUGAAUCAGGGUGGT-3′15TRPC6 siRNA 15′-UCUCUCCAUUUGGUAUGAGAAUCUU-3′5′-AAGAUUCUCAUACCAAUGGAGAGA-3′16TRPC6 siRNA 25′-CCCAAGGAAUAUUUGUUUGAGUUGA-3′5′-ACAACUCAAACAAAUAUUCCUUGGG-3′17TRPC6 siRNA 35′-GGAGCUCAGAAGAUUUCCAUCUAAA-3′5′-UUUAGAUGGAAAUCUUCUGAGCUCC-3′18TRPC7 siRNA5′-CCAUAUUCGGCUUAUCUGAAGUAAU-3′5′-AUUACUUCAGAUAAGCCCGAAUAUGG-3′ Open table in a new tab Western Blotting—HaCaT cells and hPKs were harvested by centrifugation (800 × g, 5 min, room temperature). The cells were resuspended in lysis buffer (50 mm Tris/HCl, 2 mm dithiothreitol, 0.2 μm benzamidine, 1 mm EDTA, pH 8.0) and homogenized by shearing through 26-gauge needles. After removal of nuclei (800 × g, 2 min, 4 C), the supernatants were mixed with gel loading buffer (62.5 mm Tris/HCl, 10% glycerol, 5% mercaptoethanol, 2% SDS, 0.02% bromphenol blue, pH 6.8). After electrophoresis, the proteins were transferred on nitrocellulose membrane. The membrane was incubated with a blocking solution (Invitrogen) for 2 h and overnight and then probed with using specific rabbit polyclonal anti-TRPC6 (Chemicon, 1/300), mouse monoclonal anti-cytokeratin 1/10 (Chemicon, 1/200), and mouse monoclonal anti-GAPDH (Chemicon, 1/300). The antibodies were visualized by incubation with horseradish antibody conjugate. To calculate the ratio between TRPC6, cytokeratin 1/10 and GAPDH band intensities we used Image J. Histochemistry—HaCaT cells grown on glass coverslips were washed twice with phosphate-buffered saline, fixed in 4% paraformaldehyde in phosphate-buffered saline, and stained with Mayer's hematoxylin and eosin solutions. Morphological changes were analyzed by using Nikon NIS Elements AR 2.1 software. For cytospin experiments, subconfluent hPKs were incubated with SFM containing Ca2+-free medium (negative control), 2 mm Ca2+ (positive control), or 1 μm hyperforin. After 24 h the cells were trypsinized, washed twice in phosphate-buffered saline, and centrifuged onto coated microscope slides using a cytospin centrifuge (Thermo Shandon, UK). The cells were fixed with 2% formaldehyde. Subsequently the cells were stained for TRPC6 using the labeled streptavidin biotin method according to the manufacturer's instruction (DCS, Hannover, Germany). The primary polyclonal TRPC6 antibody (Chemicon) and the secondary biotinylated multi-link antibody (Dako, Denmark) were used at a dilution of 1:200. Fluorescence Measurements—The intracellular Ca2+ concentration [Ca2+]i, barium [Ba2+]i, strontium [Sr2+]i, and sodium [Na+]i measurements in single cells were carried out using the fluorescence indicators fura-2-AM or SBFI-AM in combination with a monochromator-based imaging system (T.I.L.L. Photonics, Martinsried, Germany or Attofluor Ratio Vision System) attached to an inverted microscope (Axiovert 100; Carl Zeiss, Oberkochen, Germany). For [Ca2+]i measurements HaCaT cells and hPKs were loaded with 4 μm fura-2-AM (Invitrogen) and 0.01% Pluronic F-127 (Invitrogen) for 30 min at room temperature in a standard solution composed of 138 mm NaCl, 6 mm KCl, 1 mm MgCl2, 2 mm CaCl2, 5.5 mm glucose, and 10 mm HEPES (adjusted to pH 7.4 with NaOH). The coverslips were then washed in this buffer for 20 min and mounted in a perfusion chamber on the microscope stage. To measure Ba2+ and Sr2+ influx, the cells were incubated with Ca2+-free standard solution. The influx of Ba2+ and Sr2+ in HaCaT cells was evaluated in fura-2-loaded cells by measuring the fluorescence of Ba2+/Sr2+ fura complexes. [Na+]i concentration was measured by incubating HaCaT cells with the fluorescence dye SBFI-AM (10 μm) and 0.01% Pluronic F-127 for 40 min at room temperature in a sodium-free medium (3 mm KCl, 2 mm MgCl, 5 mm Tris, 10 mm glucose; the sodium replaced by an equimolar amount of sucrose; pH adjusted with HCl to 7.4). After washing out the fluorescence dye, sodium-containing medium (140 mm Na+) was added. For all of the fluorescence experiments, fluorescence was excited at 340 and 380 nm. After correction for background fluorescence, the fluorescence ratio F340/F380 was calculated. In all of the experiments, transfected cells (5–10 cells) of the whole field of vision were identified by their YFP fluorescence at an excitation wavelength of 480 nm. Electrophysiology—Currents in HaCaT cells were recorded in the perforated patch configuration with amphotericin B. The experiments were performed at room temperature using a Axopatch 200B amplifier (Axon Instruments). Patch pipettes of 3–5 MOhm were fabricated from borosilicate glass capillaries. The bath solution consisted of 6 mm KCl, 134 mm NaCl, 1 mm MgCl2, 2 mm CaCl2, 10 mm HEPES, 10 mm d-Glucose, 40 mm d-mannitol (pH 7.4, NaOH). The pipette solution contained 134 mm Cs-MES, 6 mm KCl, 10 mm NaCl, 1 mm MgCl2, 0.1 mm EGTA, 10 mm HEPES (pH 7.2, CsOH). Amphotericin B (Sigma) were dissolved in dimethyl sulfoxide and diluted into the pipette solution to give a final concentration of 250 μg/ml. Perforation started shortly after seal formation and reached a steady-state level within 5–10 min. The currents were recorded from holding potentials of –40 mV during linear voltage ramps at 0.67 V/s from –100 mV to +100 mV applied each 15 s. The average capacitance of the cells was 30.7 ± 1.4 pF (n = 39). Patch pipettes of 3–5 MΩ were fabricated from borosilicate glass capillaries. The experiments were analyzed using Clampfit software (Axon Instruments). The data are presented as the means ± S.E. Proliferation Measurement—Quantification of cell proliferation was determined by a nonisotopic immunoassay kit (Calbiochem, Germany), based on the measurement of bromodeoxyuridine incorporation during DNA synthesis. The assay was carried out according to the product instruction manual. MTT Assay—Estimation of cytotoxicity of hyperforin on cell viability was determined by means of MTT assay, on HaCaT keratinocytes grown on 96-well plates, after 48 h of treatment. According to the manufacturing instructions (Roche Applied Science), MTT reagent was added at a final concentration of 1 mg/ml. Incubation was continued for another 2 h, and the formazan crystals were then solubilized by 100 μl of a 20% SDS/50% N,N-dimethyl-formamide solution. After complete 12 h of solubilization, the absorption was measured at 550 nm with a correction wavelength of 620 nm using an enzyme-linked immunosorbent assay micro plate reader. Statistics—In addition to Microsoft Office Excel, GraphPad PRISM™ (version 3.0) was used for statistical analyses and to create the graphs. For statistical analyses, an unpaired Student's t test (two-tailed) was used. The data are expressed as the means ± S.D. Hyperforin Induces Differentiation in HaCaT Keratinocytes—Because of the unclear situation of TRPC channels expressed in keratinocytes, we performed RT-PCR analyses from RNA extracted from primary keratinocytes and HaCaT cells. The detection of TRPC6-encoding mRNA in both cell types provided the rationale to use hyperforin as pharmacological tools to unravel the mechanism of keratinocyte differentiation induced by high (2 mm) extracellular calcium (high [Ca2+]o), which has been shown to be an endogenous trigger (1Yuspa S.H. Kilkenny A.E. Steinert P.M. Roop D.R. J. Cell Biol. 1989; 109: 1207-1217Crossref PubMed Scopus (510) Google Scholar, 20Bikle D.D. Ratnam A. Mauro T. Harris J. Pillai S. J. Clin. Investig. 1996; 97: 1085-1093Crossref PubMed Scopus (180) Google Scholar). In the initial experimental set up, we compared cell morphology as read out in hyperforin-treated cells and cells exposed to high [Ca2+]o (Fig. 1A). Control HaCaT keratinocytes and hPKs are predominantly polygon-shaped, with clear boundaries and a large nucleus/cytoplasm ratio (Fig. 1A, left panel). After a period of 3 days in the presence of high [Ca2+]o, the cells undergo visible morphological changes, from large and flat to closely associated, elongated cells with spider web morphology (Fig. 1A, middle panel). As shown in Fig. 1A (right panel) for HaCaT cells, hyperforin induced morphological changes in HaCaT and hPK cells (data not shown), which are comparable with the morphology of cells cultured in the presence of high [Ca2+]o (compare middle and right panels). To test whether or not these morphological changes can also be detected by biochemical methods, we analyzed protein extracts of hyperforin- and high [Ca2+]o-treated cells (Fig. 1B). Western blot analyses revealed that HaCaT and hPK cells express basal levels of K1 and K10 being up-regulated during high [Ca2+]o or hyperforin treatment. K1 and K10 represent early differentiation markers, whereas IVL or transglutaminase I are late markers of keratinocyte differentiation. Using RT-PCR, we analyzed early and late differentiation markers in HaCaT (Fig. 1C) and hPK (Fig. 1D) cells. As shown in Fig. 1C low K1, K10, as well as IVL and TGM1 mRNA concentrations were detected in medium containing 0.1 mm Ca2+, whereas their mRNA levels were increased in cells cultured in the presence of either high [Ca2+]o or hyperforin. Quantification of the RT-PCR signals obtained from mRNA extracted from HaCaT (Fig. 1E) and hPKs (Fig. 1F) by normalization clearly show that incubation in the presence of high [Ca2+]o as well as hyperforin increased the transcription of early and late keratinocyte differentiation markers. Hyperforin Inhibits Proliferation in HaCaT Keratinocytes—In addition to differentiation, proliferation of keratinocytes is also controlled by intracellular free Ca2+ concentration. Therefore, we performed proliferation measurements with the bromodeoxyuridine immunoassay kit (Chemicon). Synchronized HaCaT keratinocytes incubated with high [Ca2+]o for 3 days showed significantly reduced proliferation (Fig. 2A). Notably, hyperforin (1 μm) also inhibited the proliferation of keratinocytes, as shown in Fig. 2A. To confirm these findings, we analyzed the expression of the nuclear proliferation marker protein Ki-67 by Western blotting. Ki-67 is expressed in cells undergoing the S/G2/M transition and serves as a well established marker to determine proliferating cells (21Gerdes J. Lemke H. Baisch H. Wacker H.H. Schwab U. Stein H. J. Immunol. 1984; 133: 1710-1715PubMed Google Scholar). As shown in Fig. 2B, protein expression of Ki-67 is similarly reduced in HaCaT cells treated either with hyperforin or high [Ca2+]o. To exclude toxic effects induced by hyperforin, we performed MTT assay (
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