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Expression of Human Cystatin A by Keratinocytes Is Positively Regulated via the Ras/MEKK1/MKK7/JNK Signal Transduction Pathway but Negatively Regulated via the Ras/Raf-1/MEK1/ERK Pathway

2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês

10.1074/jbc.m102021200

ISSN

1083-351X

Autores

Hidetoshi Takahashi, Masaru Honma, Akemi Ishida‐Yamamoto, Kazuhiko Namikawa, Hiroshi Kiyama, Hajime Iizuka,

Tópico(s)

melanin and skin pigmentation

Resumo

Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form ofras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1,MEK1, ERK1, ERK2, or wild-typeMEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1,ERK1, ERK2, or dominant negative forms ofMEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (−272 to −278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, orp38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK. Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent expressed in the upper epidermis. We previously reported that a potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate, increases human cystatin A expression by the activation of AP-1 proteins. Here, we delineate the signaling cascade responsible for this regulation. Co-transfection of the cystatin A promoter into normal human keratinocytes together with a dominant active form ofras increased the promoter activity by 3-fold. In contrast, a dominant negative form of ras suppressed basal cystatin A promoter activity. Further analyses disclosed that transfection of dominant negative forms of raf-1,MEK1, ERK1, ERK2, or wild-typeMEKK1 all increased cystatin A promoter activity in normal human keratinocytes, whereas wild-type raf-1,ERK1, ERK2, or dominant negative forms ofMEKK1, MKK7, or JNK1 suppressed the promoter activity. The increased or decreased promoter activity reflected the expression of cystatin A on mRNA and protein levels. These effects were not observed when a cystatin A promoter with a T2 (−272 to −278) deletion was used. In contrast, transfection of dominant negative forms of MKK3, MKK4, orp38 did not affect cystatin A promoter activity. Immunohistochemical analyses revealed that phosphorylated active extracellular signal-regulated kinases and c-Jun N-terminal kinase were expressed in the nuclei of basal cells and cells in the suprabasal-granular cell layer, respectively. These results indicate that the expression of cystatin A is regulated via mitogen-activated protein kinase pathways positively by Ras/MEKK1/MKK7/JNK and negatively by Ras/Raf/MEK1/ERK. cornified cell envelope 12-O-tetradecanoylphorbol-13-acetate protein kinase C mitogen-activated protein kinase MAPK kinase extracellular signal-regulated kinase c-Jun N-terminal kinase normal human keratinocyte chloramphenicol acetyltransferase wild type dominant negative dominant active During their migration from the basal cell layer to the horny cell layer of the skin, keratinocytes cease proliferation. They then undergo terminal differentiation, which is characterized by the production of a highly insoluble rigid structure termed cornified cell envelope (CE)1 beneath the plasma membrane (1Hohl D. Dermatologica (Basel). 1990; 180: 201-211Crossref PubMed Scopus (173) Google Scholar, 2Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. J. Invest. Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar, 3Ishida-Yamamoto A. Iizuka H. Exp. Dermatol. 1998; 7: 1-10Crossref PubMed Scopus (108) Google Scholar). Transglutaminase enzymes catalyze the assembly of this structure via formation of ε-(γ-glutamyl)lysine bonds between envelope precursors. The proteins that have been identified as constituents of the CE include involucrin (4Eckert R.L. Green H. Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (321) Google Scholar), loricrin (5Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. J. Biol. Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar), small proline-rich protein(s) (7Kartasova T. van de Putte P. Mol. Cell. Biol. 1998; 8: 2038-2195Google Scholar), elafin (8Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar), envoplakin (9Ruhrberg C. Hajibagheri M.A. Simon M. Dooley T.P. Watt F.M. J. Cell Biol. 1996; 134: 715-729Crossref PubMed Scopus (152) Google Scholar), desmosomal components (10Ishida-Yamamoto A. Kartasova T. Matsuo S. Kuroki T. Iizuka H. J. Invest. Dermatol. 1997; 108: 12-16Abstract Full Text PDF PubMed Scopus (34) Google Scholar), and plasminogen-activator inhibitor 2 (10Ishida-Yamamoto A. Kartasova T. Matsuo S. Kuroki T. Iizuka H. J. Invest. Dermatol. 1997; 108: 12-16Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Recent evidence indicates that involucrin is an early component of CE and provides a scaffold onto which other precursor proteins are incorporated (8Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 10Ishida-Yamamoto A. Kartasova T. Matsuo S. Kuroki T. Iizuka H. J. Invest. Dermatol. 1997; 108: 12-16Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Cystatin A, a cysteine proteinase inhibitor, belongs to the cystatin superfamily. In the epidermis cystatin A is expressed in the upper spinous to granular cell layers (12Lobitz C.J. Buxman M.M. J. Invest. Dermatol. 1982; 78: 150-154Abstract Full Text PDF PubMed Scopus (23) Google Scholar). In addition to its proteinase inhibitory effect, cystatin A is known to be an early precursor protein of CE in keratinocytes (8Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). The human cystatin A gene is located on 3q21 (13Hsieh W.-T. Fong D. Sloane B.F. Golembieski W. Smith D.I. Genomics. 1991; 9: 207-209Crossref PubMed Scopus (22) Google Scholar) and consists of three exons that are separated by 14- and 3.6-kilobase introns, respectively (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). A potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induces terminal differentiation of keratinocytes (15Hawley-Nelson P. Roop D.R. Cheng C.K. Krieg T.M. Yuspa S.H. Mol. Carcinog. 1998; 1: 202-211Crossref Scopus (50) Google Scholar, 16Mufson R.A. Steinberg M.L. Defendi V. Cancer Res. 1982; 42: 4600-4605PubMed Google Scholar). Recent studies revealed that TPA increases cystatin A expression at both the mRNA and protein levels (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). The 5′-flanking region of the cystatin A gene contains at least two putative TPA-responsive regions, T1 (−189 to −196) and T2 (−272 to −278). The AP-1 proteins, c-Jun, c-Fos, and Jun D, regulate cystatin A promoter activity via PKC activation (8Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). However, the precise regulatory mechanism of AP-1-dependent cystatin A expression in keratinocytes remains to be determined. Cell growth, differentiation, and apoptosis are mediated by the activation of mitogen-activated protein kinase (MAPK) pathways. A MAPK is activated by a specific MAPK kinase (MAPKK) through the phosphorylation of specific threonine and tyrosine residues (Thr-X-Tyr) in MAPK. MAPKK is activated by MAPKK kinase through the phosphorylation of specific threonine and tyrosine residues (Thr-Y-Tyr) in MAPKK (17Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 18Zettergren C.J. Peterson L. Wuepper K.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 238-242Crossref PubMed Scopus (85) Google Scholar). These kinases constitute MAP kinase cascades. In mammalian cells, at least four MAPKs, namely, extracellular signal-regulated kinases (ERKs) (19Cobb M.H. Robbins D.J. Boulton T.G. Curr. Opin. Cell Biol. 1991; 3: 1025-1032Crossref PubMed Scopus (120) Google Scholar), c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs) (20Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar), p38 (21Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1507) Google Scholar), and ERK5/big MAP kinase have been identified (22Abe J. Kusuhara M. Ulevitch R.J. Berk B.C. Lee J.D. J. Biol. Chem. 1996; 271: 16586-16590Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Although the ERKs are usually activated by mitogenic stimuli, JNK and p38 are activated by other stimuli such as environmental stress, ultraviolet irradiation, osmotic pressure, and various cytokines including tumor necrosis factor α and transforming growth factor β (23Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar, 24Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). ERK/big MAP kinase 5 seems to play a significant role in epidermal growth factor-induced cell proliferation (22Abe J. Kusuhara M. Ulevitch R.J. Berk B.C. Lee J.D. J. Biol. Chem. 1996; 271: 16586-16590Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Recent analyses indicate that TPA-induced gene expression of keratinocytes is associated with MAP kinase activation. For example, the expression of the human involucrin gene is regulated by MAPK cascades (25Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In the present study, we have analyzed the regulatory pathway(s) of various MAPK-signaling systems that control cystatin A expression of normal human keratinocytes. Normal human keratinocytes (NHK cells) were obtained during plastic surgery. Informed consent was obtained from the patients. The cells were cultured in keratinocyte growth medium containing epidermal growth factor (10 ng/ml), insulin (5 μg/ml), and bovine pituitary extract (50 μg/ml) at 37 °C in 5% CO2 in air. NHK cells were maintained in a subconfluent state by subculturing every 4–5 days. Cells were seeded into 60-mm diameter plastic dishes at concentrations of 4 × 105cells/5 ml. We previously have published the structure of the cystatin A promoter construct (+77 to −648) linked to the chloramphenicol acetyltransferase (CAT) gene (p648CAT) (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The structure of p648ΔT1 and p648ΔT2, with the deleted TPA-responsive elements T1 (−189 to −196) and T2 (−272 to −278), respectively, has been described previously (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Wild-type (wt)ERK1, wt-ERK 2, dominant negative (dn)-ERK1 (K71R), and dn-ERK2 (K52R), each cloned in pCEP, were provided kindly by Dr. Melanie Cobb (26Robbins D.J. Zhen E Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 68: 5097-5106Abstract Full Text PDF Google Scholar). Dominant negative Ha-ras (S17W) (dn-ras) and dn-MEKK1, cloned in pSRα, and wt-Raf-1 and dn-raf-1 (K375W), each cloned in pRSV, were provided kindly by Dr. Michael Karin (27Minden A. In A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). Constitutively active ras (G12V) cloned in pZipNeoSV(X)1 (da-ras) was a kind gift of Dr. Michael Simonson (28Herman W.H. Simonson M.S. J. Biol. Chem. 1995; 270: 11654-11661Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). wt-MEKK1, dn-MEK1, and dn-MKK4 (S220A), each cloned in pEECMV, were provided kindly by Dr. Dennis Templeton (29Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar,30Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar). Dominant negative MKK7 (MKK7KL) cloned in pSRα (dn-MKK7) was a kind gift of Dr. Eisuke Nishida (31Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). Dominant negative MKK3 (MKK3Ala) cloned in pRSV (dn-MKK3), dominant negative p38 MAPK (p38AGF) cloned in pCMV5 (dn-p38), and dominant negativeJNK1 (JNK1APF) cloned in pcDNA3 (dn-JNK) were provided kindly by Dr. Roger Davis (20Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar, 32Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar, 33Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1150) Google Scholar). The β-galactosidase expression vector was provided kindly by Dr. Takeshi Watanabe (Medical Institute of Bioregulation, Kyushu University, Japan). The transfection of plasmid DNA into cells was performed by the liposome method using Lipofectin (34Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7413-7417Crossref PubMed Scopus (4388) Google Scholar). Typically, 5 μg of reporter plasmid and 2 μg of β-galactosidase plasmid were co-transfected with 2 μg of various expression vectors or empty vector into 1 × 105 NHK cells. The β-galactosidase plasmid was used as the internal standard to normalize the efficiency of each transfection. After 48 h, the cells were collected, and CAT assays were performed (35Neuman J.R. Morency C.A. Russian K.D. BioTechniques. 1987; 5: 441Google Scholar). The enzyme activity of β-galactosidase in the transfected cell extracts was measured spectrophotometrically (36Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Relative CAT activities were expressed as the fold increase in the acetylated fraction after correction for the activity of the 0-CAT vector. The cDNA of dominant negative mutants of extracellular signal-regulated kinase (dn-REK1, human REK1 K52R) and dn-ERK2 (rat ERK2 K71R) were provided by Dr. Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX) (26Robbins D.J. Zhen E Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 68: 5097-5106Abstract Full Text PDF Google Scholar). Recombinant adenoviruses (constitutively active ras (G12V) (Ad-da-ras) and Ad-dn-ERK1) were prepared using the procedures described previously (37Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). Briefly, cDNA fragments encoding these proteins were cloned into an expression cosmid cassette designated pAxCAwt (38Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (788) Google Scholar). This was created from the human type 5 adenovirus genome from which the E1A, E1B, and E3 regions had been deleted and replaced with the appropriate expression unit under the control of the CAG promoter (39Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4617) Google Scholar). Each expression cosmid cassette and EcoT221-digested adenovirus DNA terminal protein complex (Ad5-dIX DNA-TPC) was co-transfected into human embryonic kidney (HEK)293 cells, and the recombinant adenoviruses were generated by homologous recombination and amplified in HEK293 cells. Moreover, high titers of recombinant viral stocks were generated in HEK293 cells, purified by cesium gradient centrifugation (40Kanegae Y. Makimura M. Saito I. Jpn. J. Med. Sci. Biol. 1994; 47: 157-166Crossref PubMed Scopus (431) Google Scholar), and stored at −80 °C until use. The viral titers were determined by plaque-forming assays in HEK293 cells. AxCAdaMEK, representing constitutively active human MEK1 (termed dominant active MEK1 or da-MEK1), without the nuclear export signal was constructed as described elsewhere (37Namikawa K. Honma M. Abe K. Takeda M. Mansur K. Obata T. Miwa A. Okado H. Kiyama H. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). The AxCANLacZ was provided kindly by Dr. Izumi Saito (University of Tokyo) (41Terashima T. Miwa A. Kanegae Y. Saito I. Okado H. Anat. Embryol. 1997; 196: 363-382Crossref PubMed Scopus (41) Google Scholar). The cells were infected by adding recombinant adenovirus (multiplicity of infection, 10) to keratinocyte growth medium. The cells were incubated at 37 °C for 60 min. The medium was changed to fresh keratinocyte growth medium, and the cells were incubated at 37 °C for 24 h. NHK cells (5 × 106) were homogenized using the 6 mguanidine-cesium-guanidine method (36Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total RNA (30 μg) was electrophoresed in 1% agarose-formaldehyde gels and transferred to nylon filters. Human cystatin A and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were labeled with [32P]dCTP by the random primer method. Hybridization was performed as described previously (36Maniatis T. Fritsch E.H. Sambrook T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). GAPDH cDNA was a generous gift of Dr. Michael Tainsky (M.D. Anderson Cancer Center, University of Texas, Houston, TX). Total RNA was extracted from NHK cells and transcribed into cDNA as described previously (42Takahashi H. Kinouchi M. Tamura T. Iizuka H. Br. J. Dermatol. 1996; 134: 1065-1069Crossref PubMed Scopus (31) Google Scholar). cDNA was synthesized from 3 μg of total RNA in a 30-μl reaction mixture using random primers. Real-time polymerase chain reaction was performed according to the protocol of the LightCycler-DNA Master SYBER Green 1 kit (Roche Diagnostics K. K., Mannheim, Germany). Human cystatin A was amplified using specific oligonucleotide primers (5′-ATGATACCTGGAGGCTTATCT-3′ and 5′-CAAGTCCTCATTTTGTCCGGG-3′). The housekeeping gene GAPDH as a standard was amplified using the specific oligonucleotides 5′-TGGGCTACACTGAGCACCAG-3′ and 5′-CAGCGTCAAAGGTGGAGGAG-3′ (43Torma H. Karlsson T. Michaelsson G. Rollman O. Vahlquist A. Acta Derm. Venereol. 2000; 80: 4-9Crossref PubMed Scopus (45) Google Scholar). DNA was amplified for 40 cycles on a LightCycler (Roche Diagnostics K.K.) at 95 °C for 5 s, 65 °C for 5 s, and 72 °C for 5 s. Cell extracts were prepared according to the method of Zettergren et al. (18Zettergren C.J. Peterson L. Wuepper K.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 238-242Crossref PubMed Scopus (85) Google Scholar). Cytosolic extracts (30 μg of protein) were electrophoresed on 15% SDS-polyacrylamide gel electrophoresis gels and electroblotted onto nitrocellulose for 1 h in a buffer containing 25 mmTris-HCl (pH 8.3), 192 mm glycine, and 20% methanol. The blots were blocked with 5% nonfat milk in PBS for 1 h at room temperature and were then incubated at 4 °C overnight with anti-human cystatin A antibody (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar), anti-MEKK1 antibody (New England Biolabs), anti-MKK3 antibody (New England Biolabs), anti-MKK4 antibody (Santa Cruz Biotechnology, Inc.), anti-MKK7 antibody (Santa Cruz Biotechnology, Inc.), anti-JNK antibody (New England Biolabs), anti-ERK antibody (New England Biolabs), or anti-p38 antibody (New England Biolabs) diluted 500-fold in Tris-buffered saline (pH 7.6). After washing with 0.1% Tween 20 in Tris-buffered saline at room temperature, immunodetection was performed using a blot detection kit for rabbit antibody (Amersham Pharmacia Biotech). Tissue specimens were fixed in 10% formalin, embedded in paraffin, and cut into consecutive sections of 5-μm thickness. The sections were treated with 0.3% hydrogen peroxide to block endogenous peroxidase activity. After incubation with normal goat serum diluted in PBS for 10 min, the sections were incubated for 4 h at room temperature with anti-ERK, anti-phospho-ERK, anti-JNK, or anti-phospho-JNK (New England Biolabs) antibodies diluted 1:200 in PBS. The sections were then washed with PBS followed by staining with the Histofine streptavidin-biotin (SAB)-PO(M) kit (Nichirei, Tokyo, Japan). Biotinylated goat anti-rabbit antibody was used as the secondary antibody, and the immune reaction was visualized by avidin/biotin complexing with 0.03% hydrogen peroxide as a substrate and diaminobenzidine as a chromogen. The sections were then counterstained with hematoxylin and mounted in PermaFlurTMaqueous mounting medium (IMMUNON, Pittsburgh, PA). DMCB153 medium was purchased from Life Technologies, Inc. Penicillin and streptomycin were obtained from M.A. Bioproducts (Walkersville, MD). PD90859 and SB203580 were purchased from Calbiochem (San Diego, CA). [α-32P]dCTP was obtained from Amersham Pharmacia Biotech. All other chemicals were purchased from Nakarai Chemicals Ltd. (Kyoto, Japan). Ras is a GTP-binding protein located upstream of the MAP kinase cascade (17Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 18Zettergren C.J. Peterson L. Wuepper K.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 238-242Crossref PubMed Scopus (85) Google Scholar). Although the activation of Ras is usually associated with receptor tyrosine kinase activation, recent studies demonstrated that Ras is also a downstream target of PKC (44Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (687) Google Scholar, 45Marais R. Light Y. Paterson H.F. Mason C.S. Marshall C.J. J. Biol. Chem. 1997; 272: 4378-4383Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). To determine whether Ras is involved in TPA-stimulated cystatin A promoter activity, the promoter vector (p648CAT) was transfected into NHK cells together with either da-ras or dn-ras vectors. As reported previously (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), TPA significantly stimulated cystatin A promoter activity (Fig. 1). Co-transfection of the da-ras vector with p648CAT increased cystatin A promoter activity to a similar extent as TPA. The stimulatory effect of da-Ras was not affected by the presence or absence of TPA (Fig. 1), suggesting that the effect of the latter on cystatin A promoter activity is mediated via the activation of Ras. In contrast, co-transfection of dn-ras decreased both basal and TPA-stimulated promoter activity. This is consistent with the idea that TPA mediates its effects through the activation of Ras and also suggests that Ras is partially activated in NHK cells under the conditions employed (i.e. with 0.01 mm calcium). Ras activates various MAPK cascades including Raf-1/MEK1/ERK and MEKK1/MKK3/4/6/7/p38/JNK pathways (17Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2286) Google Scholar, 18Zettergren C.J. Peterson L. Wuepper K.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 238-242Crossref PubMed Scopus (85) Google Scholar). The effect of the Raf-1/MEK1/ERK pathway on cystatin A promoter activity was analyzed. Transfection of wt-raf or wt-ERK1/2 into p648CAT-transfected NHK cells decreased basal cystatin A promoter activity (Fig.2). On the other hand, transfection of the dominant negative forms of raf-1, MEK1, orERK1/2 markedly stimulated basal cystatin A promoter activity. The stimulatory effect was similar to that of transfecting da-ras (Fig. 2). The da-ras-induced stimulation of cystatin A promoter activity was not affected by co-transfection of wt-raf-1 or wt-ERK1/2 but was augmented by co-transfection of dominant negative forms of raf-1, MEK1, orERK1/2 (Fig. 2). This suggests that the Raf-1/MEK1/ERK1/2 pathway mediates the inhibitory effect on cystatin A promoter activity in NHK cells. Consistent with this, a MEK1 inhibitor, PD90859, increased cystatin A promoter activity in a dose-dependent manner (Fig.3). This was observed even in the absence of transfection of da-Ras, again indicating that Ras is partially activated under our culture conditions. Increased promoter activity caused by PD90859 was augmented by the transfection of da-Raf-1 (Fig.3).Figure 3The MEK1 inhibitor PD98059 increases cystatin A promoter activity. Subconfluent NHK cells, which were transfected with 5 μg of p648CAT and 2 μg of da-Ras (closed box) or pSRa (empty vector, open box) were cultured for 24 h. The cells were then treated with various concentrations of PD98059 for 24 h, and the CAT activity was measured. The average CAT activities relative to the promoterless vector were calculated from at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Raf-1 is located upstream of ERK1. Accordingly, the stimulatory effect of dn-raf-1 was inhibited by the co-transfection of wt-ERK1 but not by co-transfection of dn-ERK1(Fig. 4). Co-transfection of dominant negative forms of JNK1 or p38 vectors had no effect on dn-raf-1-induced cystatin A promoter activity (Fig. 4), suggesting that the effect of dn-raf-1 is independent of JNK or p38 pathways. These results indicate that (i) the Raf-1/MEK1/ERK pathway suppresses cystatin A expression in NHK cells, (ii) Raf-1 is functionally located upstream of MEK1 and ERK1/2 in this cascade, and (iii) the Raf-1-dependent inhibitory pathway is distinct from JNK1 or p38 pathways in NHK cells. In addition to the Raf-1/MEK1/ERK pathway, Ras also activates the MEKK1-signaling cascade (46Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (847) Google Scholar). MEKK1 activates MKK4, which in turn activates JNK and p38 (27Minden A. In A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar, 29Yan M. Dai T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (660) Google Scholar). MEKK1 also activates MKK7, which is a specific activator of JNK (31Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). Transfection of wt-MEKK1 increased cystatin A promoter activity in NHK cells (Fig. 5). This effect was similar to that of da-ras, and co-transfection of both da-Ras and wt-MEKK1 had no additive effect on the increased promoter activity (Fig. 5). This indicates that da-Ras mediates its stimulatory effect through MEKK1. Further analyses disclosed that the effect of da-Ras depends on MEKK1, MKK7, and JNK1 but not on MKK4 or p38. The transfection of dn-MEKK1, dn-MKK7, or dn-JNK suppressed basal promoter activity, whereas co-transfection of da-ras had no stimulatory effect (Fig.5). Transfection of dn-MKK3, dn-MKK4, or dn-p38 had no effect on basal promoter activity either. Furthermore, da-Ras showed stimulatory effects in these cells, indicating that MKK3, MKK4, or p38 are involved in the Ras-dependent activation of cystatin A expression in NHK cells. Western blot analysis showed the expression of MKK3, MKK4, and p38 in NHK cells. The p38 inhibitor, SB203580, also failed to diminish the increased cystatin A promoter activity induced by wtMEKK1 (data not shown). As described above, MEKK1 is located downstream of active Ras. Consistent with this, the transfection of MEKK1 stimulated cystatin A promoter activity in NHK cells (Fig.6). Co-transfection with dn-MKK7 or dn-JNK inhibited MEKK1-dependent stimulation of cystatin A promoter activity. Co-transfection of wt-ERK1 inhibited the MEKK1-stimulated promoter activity, whereas dn-ERK1 and dn-MEK1 augmented it. This was expected, because the suppressive effect of the Raf-1/MEK1/ERK pathway on cystatin A expression of NHK cells was abrogated under these conditions. Co-transfection of dn-MKK3, dn-MKK4, or dn-p38 had no effect on MEKK1-stimulated cystatin A promoter activity in NHK cells (Fig. 6). Western blot analysis showed the expression of MKK3, MKK4, and p38, suggesting an independent effect for promoter activity. Real-time quantitative reverse transcription polymerase chain reaction analysis confirmed that the transfection of wtMEKK1 into NHK cells increased cystatin A mRNA levels, whereas the transfection of dn-MEKK1, dn-MEK7, or dn-JNK decreased them (Fig.7). The transfection of dn-MKK3, dn-MKK4, dn-MKK7, or dn-p38 was without effect (data not shown). These results indicate that (i) the MEKK1/MKK7/JNK pathway induces up-regulation of cystatin A promoter activity, (ii) MEKK1 is functionally located upstream of MKK7 and JNK in this cascade, and (iii) activation of the MEKK1/MKK7/JNK pathway is distinct from the inhibitory pathway that depends on Raf-1/MEK1/ERK.Figure 7Influence on cystatin A mRNA expression by transfection of wt-MEKK1, dn-MEKK1, dn-MKK7, or dn-JNK1 expression vectors into NHK cells. NHK cells were cultured under low Ca2+ (0.01 mm) conditions. Subconfluent NHK cells were transfected with 2 μg of wt-MEKK1, dn-MEKK1, dn-MKK7, or dn-JNK1 expression vectors and cultured for 24 h under low Ca2+ (0.05 mm) conditions in the presence or absence of TPA (10 ng/ml). The expression level of cystatin A mRNA in the control was designated as 1.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previous analyses revealed that the cystatin A promoter contains at least two putative TPA-responsive elements, T1 (−189 to −196) and T2 (−272 to −278), and T2 is most likely critical for TPA-dependent stimulation of cystatin A promoter activity (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To determine the critical region(s) for MAPK-dependent positive and negative regulation of cystatin A expression, T1- and/or T2-deleted constructs were co-transfected into NHK cells together with various MAPK vectors (Fig. 8). Deletion of T1 had no effect on the promoter activity; the T1-deleted vector allowed the retention of dn-MEK1-, dn-ERK1-, and wtMEKK1-dependent stimulatory effects on cystatin A promoter activity. In contrast, the T2-deleted vector resulted in a loss of stimulatory effects of dn-MEK1, dn-ERK1, and wtMEKK1 (Fig. 8). These results indicate that the T2 region of the cystatin A gene is critical for the regulation of not only the stimulatory MEKK1/MKK7/JNK pathway but also the inhibitory Raf-1/MEK1/ERK pathway. Because the Lipofectin method is not efficient enough for detecting changes in cystatin A mRNA levels by Northern blot or protein levels by Western blot in NHK cells, an adenovirus vector-dependent transfection system was utilized. Transfection of adenovirus vector containing da-ras (AxCAdaRas) increased cystatin A mRNA and protein levels (Fig. 9, lane 3). The transfection of adenovirus vector containing a dominant negative form of ERK1 (AxCAdnERK1) also increased cystatin A mRNA and protein levels (Fig. 9, lane 4). The co-transfection of AxCAdaRas and AxCAdn-ERK1 resulted in a more marked increase in cystatin A promoter activity (Fig. 9, lane 5). In contrast, the transfection of dominant negative MEKK1-expressing adenovirus vector (Ad-dn-MEKK1) suppressed the cystatin A expression. These results indicate that the activation of Ras does indeed stimulate cystatin A expression in NHK cells, which can be suppressed by the Raf-1/MEK1/ERK pathway. Immunohistochemical analyses were performed to locate active ERK and JNK proteins in the normal human epidermis. ERKs were expressed in nuclei of basal cell layers and cytoplasm of suprabasal cell layer (Fig.10A). The active phosphorylated forms of ERKs were present exclusively in the nuclei of the basal cell layer (Fig. 10B). This suggests that cystatin A expression is suppressed because of the activated Raf-1/MEK1/ERK pathway in the basal cell layer. On the other hand, JNK was detected in both cytosole and nuclei of upper spinous cell and granular cell layers (Fig. 10C). Furthermore, active phosphorylated JNK was found in the nuclei of upper spinous cell and granular cell layers (Fig.10D). This suggests that cystatin A expression is stimulated because of the activation of MEKK1/MKK7/JNK pathway as well as loss of inhibitory tonus via the Raf-1/MEK1/ERK pathway. All these results are consistent with the suprabasal expression pattern of cystatin A in the normal human epidermis (47Tezuka T. Qing J. Saheki M. Kusuda S. Takahashi M. Dermatology. 1994; 188: 21-24Crossref PubMed Scopus (36) Google Scholar). Our results clearly demonstrated that AP-1-dependent cystatin A expression in the human epidermis is regulated by two MAP kinase systems: positively via MEKK1/MKK7/JNK and negatively via Raf-1/MEK1/ERK. Although both pathways are triggered by the activation of Ras, the differential localization of downstream targets (active ERK in the basal epidermis and active JNK in the upper epidermis) explains the suprabasal expression of cystatin A in the normal human epidermis. AP-1 is comprised of Jun and Fos protein families. Previously we reported that c-Fos and c-Jun and/or Jun D are involved in cystatin A expression (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Because active JNKs efficiently phosphorylate c-Jun and less efficiently phosphorylate Jun D, but do not phosphorylate Jun B at all, it is conceivable that c-Jun and/or Jun D are the regulatory factors for cystatin A expression (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). It should be noted that c-Fos is constitutively expressed, whereas c-Jun is marginally expressed in the normal human epidermis (48Fisher G.J. Talwar H.S. Lin J. In P. McPhillips F. Wang Z.-Q. Li X. Wan Y. Kang S. Voorhees J.J. J. Clin. Invest. 1998; 101: 1432-1440Crossref PubMed Scopus (331) Google Scholar). This suggests that c-Jun is more critical than c-Fos for AP-1-dependent regulation in normal human epidermis. It is known that the phosphorylation of c-Jun by JNK reduces its susceptibility to proteasome attack, increasing its half-life (49Musti A.M. Treier M. Bohmann D. Science. 1997; 275: 400-402Crossref PubMed Scopus (410) Google Scholar). The activation of the Raf-1/MEK1/ERK pathway is usually associated with cell proliferation (19Cobb M.H. Robbins D.J. Boulton T.G. Curr. Opin. Cell Biol. 1991; 3: 1025-1032Crossref PubMed Scopus (120) Google Scholar). On the other hand, terminal differentiation of keratinocytes is associated with growth arrest associated with the activation of other MAP kinases (50Schmidt M. Goebeler M. Posern G. Feller S.M. Seitz C.S. Brocker E.B. Rapp U.R. Ludwig S. J. Biol. Chem. 2000; 275: 41011-41017Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In addition to TPA, calcium also induces keratinocyte differentiation, and our preliminary analysis suggested that high calcium concentrations (1.0 mm) decreased phosphorylated ERKs in NHK cells (data not shown). This is consistent with the finding that cystatin A expression is increased by calcium treatment in NHK cells (6Takahashi H. Kinouchi M. Wuepper K.D. Iizuka H. J. Invest. Dermatol. 1997; 108: 843-847Abstract Full Text PDF PubMed Scopus (20) Google Scholar). TPA mediates its effects through the activation of PKC. Previously we reported that the expression of cystatin A is stimulated by PKC-α (14Takahashi H. Asano K. Kinouchi M. Ishida-Yamamoto A. Wuepper K.D. Iizuka H. J. Biol. Chem. 1998; 273: 17375-17380Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Interestingly, the expression of involucrin, another CE precursor protein, is also stimulated by PKC-α and PKC-η (51Takahashi H. Asano K. Manabe A. Kinouchi M. Ishida-Yamamoto A. Iizuka H. J. Invest. Dermatol. 1998; 110: 218-223Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) as well as by calcium. Recently Ng et al. (52Ng D.C. Shafaee S. Lee D. Bikle D.D. J. Biol. Chem. 2000; 275: 24080-24088Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) reported that the calcium-responsive region of the involucrin gene is regulated by AP-1. Although these effects suggest that the expression of cystatin A and involucrin is regulated by a similar mechanism, Efimova et al. (25Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) reported that involucrin gene expression in keratinocytes is stimulated by the PKC/Ras/MEKK1/p38 pathway but not by the JNK pathway. This implicates distinctive MAP kinase-dependent regulatory mechanisms among the keratinocyte differentiation markers. In the present study, we have characterized the nature of cystatin A expression in keratinocytes. The results demonstrate that (i) the stimulatory effect of TPA on cystatin A expression is mediated through Ras, (ii) the Ras/MEKK1/MEK7/JNK/AP1 and Raf-1/MEK1/ERK pathways mediate stimulation and inhibition of cystatin A promoter activity, respectively, and (iii) these pathways depend on an intact T2 region (−272 to −278) of the cystatin A gene. Our results define another novel TPA-dependent MAP kinase-mediated regulatory mechanism for keratinocyte differentiation. The technical assistance of K. Nishikura, K. Takahashi, and Y. Mera and the secretarial assistance of Y. Maekawa are greatly appreciated.

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