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• W2042180438 abstract "DAX-1 and SF-1 are members of the orphan nuclear receptor superfamily that are critical regulatory components of the hypothalamic-pituitary-adrenal-gonadal axis. In adrenal and gonadal tissues they regulate the expression of the cytochrome P450 steroid hydroxylase genes, key mediators of steroidogenesis. The identification of a number of steroid hydroxylases in human skin prompted us to investigate the presence of DAX-1 and SF-1. Immuno histochemical analysis of human skin revealed a distinctive staining pattern for DAX-1 and SF-1 in skin and its appendages. Prominent staining for DAX-1 was confined to the epidermis, sebaceous glands, sweat glands, and outer root sheath of the hair follicle with weaker expression in the inner root sheath, matrix cells, and dermal papilla cells. Similarly, SF-1 was also detected in the epidermis but displayed a scattered nuclear pattern across all layers. SF-1 immunoreactivity was also detected in the exocrine glands and was stronger than DAX-1 in the inner root sheath, matrix cells, and dermal papilla cells. Co-localization of DAX-1 and SF-1 was demonstrated by immunocytochemistry in the HaCaT keratinocyte cell line, primary keratinocytes, preadipocytes, and dermal papilla cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated the expression of DAX-1 and SF-1 mRNA in whole human skin and Western analysis also confirmed the presence of DAX-1 protein in skin-derived cells. Our investigations demonstrate that two important regulators of steroidogeneisis are present in human skin and its appendages. These transcription factors may have a role in cutaneous steroidogenesis and thus be involved in hair follicle cycling or pathologies associated with steroids. Further studies are needed to determine the functional roles of DAX-1 and SF-1 in human skin. DAX-1 and SF-1 are members of the orphan nuclear receptor superfamily that are critical regulatory components of the hypothalamic-pituitary-adrenal-gonadal axis. In adrenal and gonadal tissues they regulate the expression of the cytochrome P450 steroid hydroxylase genes, key mediators of steroidogenesis. The identification of a number of steroid hydroxylases in human skin prompted us to investigate the presence of DAX-1 and SF-1. Immuno histochemical analysis of human skin revealed a distinctive staining pattern for DAX-1 and SF-1 in skin and its appendages. Prominent staining for DAX-1 was confined to the epidermis, sebaceous glands, sweat glands, and outer root sheath of the hair follicle with weaker expression in the inner root sheath, matrix cells, and dermal papilla cells. Similarly, SF-1 was also detected in the epidermis but displayed a scattered nuclear pattern across all layers. SF-1 immunoreactivity was also detected in the exocrine glands and was stronger than DAX-1 in the inner root sheath, matrix cells, and dermal papilla cells. Co-localization of DAX-1 and SF-1 was demonstrated by immunocytochemistry in the HaCaT keratinocyte cell line, primary keratinocytes, preadipocytes, and dermal papilla cells. Reverse transcriptase-polymerase chain reaction analysis demonstrated the expression of DAX-1 and SF-1 mRNA in whole human skin and Western analysis also confirmed the presence of DAX-1 protein in skin-derived cells. Our investigations demonstrate that two important regulators of steroidogeneisis are present in human skin and its appendages. These transcription factors may have a role in cutaneous steroidogenesis and thus be involved in hair follicle cycling or pathologies associated with steroids. Further studies are needed to determine the functional roles of DAX-1 and SF-1 in human skin. dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 steroidogenic factor-1 The cytochrome P450 steroid hydroxylases are a group of enzymes predominantly located in the adrenal cortex and gonads, where they act in a sequential cascade converting cholesterol, the common precursor of steroid hormones, into biologically active steroids. The steroid hormones synthesized by these enzymes are involved in a plethora of physiologic and developmental processes, including ion balance, metabolism, sexual development, and reproductive function. Studies of several mouse steroid hydroxylase promoters led to the identification and cloning of an orphan nuclear receptor, steroidogenic factor 1 (SF-1) (Lala et al., 1992Lala D.S. Rice D.A. Parker K.L. Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi taruzu-factor I.Mol Endocrinol. 1992; 6: 1249-1258Crossref PubMed Scopus (511) Google Scholar), also known as adrenal 4 binding protein (Ad4BP) (Honda et al., 1993Honda S. Morohashi K. Nomura M. Takeya H. Kitajima M. Omura T. Ad4 BP regulating steroidogenic P450 gene is a member of steroid hormone receptor superfamily.J Biol Chem. 1993; 268: 7494-7502Abstract Full Text PDF PubMed Google Scholar). It was found to induce the expression of all the steroid hydroxylases by binding as a monomer to consensus estrogen receptor half site (AGGTCA) regulatory elements in their gene promoters. Disruption of Ftzf1, the gene that encodes murine SF-1, causes adrenal gland and gonadal deficiency accompanied by prominent XY sex reversal (Luo et al., 1994Luo X. Ikeda Y. Parker K.L. A cell specific nuclear receptor is required for adrenal development and for male sexual differentiation.Cell. 1994; 77: 481-490Abstract Full Text PDF PubMed Scopus (1311) Google Scholar), which is consistent with the phenotype recently detected in a female XY human who had a heterozygous mutation in the FTZ-F1 gene (Achermann et al., 1999Achermann J.C. Ito M. Hindmarsh P.C. Jameson J.L. A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans.Nature Genet. 1999; 22: 125-126Crossref PubMed Scopus (519) Google Scholar). These studies demonstrated the critical role that SF-1 plays in the development and function of these endocrine organs. SF-1 has also been found to regulate the expression of other components of the hypothalamic-pituitary-gonadal-adrenal axis such as the adrenocorticotrophic hormone receptor (ACTH-R), the gonadotrophin-releasing hormone receptor (GnRH-R), the β-subunit of luteinizing hormone (LHβ) and follicle stimulating hormone β-subunit (FSHβ), the cholesterol transporter steroidogenic acute regulatory (StAR) protein, and Müllerian inhibiting substance, a member of the transforming growth factor β family (reviewed inMorohashi, 1999Morohashi K. Gonadal and extragonadal functions of Ad4 BP/SF-1. Developmental aspects.TEM. 1999; 10: 169-173Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In addition, SF-1 is important for the regulation of the transcriptional repressor, dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1) (Burris et al., 1995Burris T.P. Guo W. Le T. McCabe R.B. Identification of a putative steroidogenic factor-1 response element in the DAX-1 promoter.Biochem Biophys Res Commun. 1995; 214: 576-581Crossref PubMed Scopus (92) Google Scholar;Kawabe et al., 1999Kawabe K. Shikayama T. Tsuboi H. et al.Dax-1 as one of the target genes of AD 4 BP/SF-1.Mol Endocrinol. 1999; 13: 1267-1284Crossref PubMed Scopus (101) Google Scholar), which was recently found to bind RNA and shuttle between nucleus and cytoplasm (Lalli et al., 2000Lalli E. Ohe K. Hindelang C. Sassone-Corsi P. Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA.Mol Cell Biol. 2000; 20: 4910-4921Crossref PubMed Scopus (104) Google Scholar). Mutations in DAX-1 were found to cause X-linked adrenal hypoplasia congenita (AHC) and the associated hypogonadotropic hypogonadism (HHG) (Muscatelli et al., 1994Muscatelli F. Strom T.M. Walker A.P. et al.Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.Nature. 1994; 372: 672-676Crossref PubMed Scopus (599) Google Scholar), demonstrating that DAX-1, like SF-1, is also crucial for adrenal and gonadal development. DAX-1 shares a similar tissue distribution with SF-1 and has been shown to interact directly with SF-1 to repress its transcriptional activity (Ito et al., 1997Ito M. Yu R. Jameson J.L. DAX-1 inhibits SF-1- mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita.Mol Cell Biol. 1997; 17: 1476-1483Crossref PubMed Scopus (377) Google Scholar;Zazopoulos et al., 1997Zazopoulos E. Lalli E. Stocco D.M. Sassone-Corsi P. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis.Nature. 1997; 390: 311-315Crossref PubMed Scopus (352) Google Scholar). DAX-1 is also able to block the transcriptional activity of other proteins involved in steroidogenesis and thus impede the steroidogenic process. One such regulated gene includes StAR, a protein that mediates the first step of steroidogenesis by transporting extramitochondrial cholesterol into the inner mitochondrial membrane for conversion into pregnenolone by CYP11A1 (Stocco and Sodeman, 1991Stocco D.M. Sodeman T.C. The 30 kDa mitochondrial proteins induced by hormone stimulation in MA-10 mouse Leydig tumour cells are processed from larger precursors.J Biol Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar;Zazopoulos et al., 1997Zazopoulos E. Lalli E. Stocco D.M. Sassone-Corsi P. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis.Nature. 1997; 390: 311-315Crossref PubMed Scopus (352) Google Scholar;Reinhart et al., 1999Reinhart A.J. Williams S.C. Stocco D.M. Transcriptional regulation of the StAR gene.Mol Cell Endocrinol. 1999; 151: 161-169Crossref PubMed Scopus (74) Google Scholar). In addition to steroidogenic tissues,Asa et al., 1996Asa S.L. Bamberger A. Cao B. Wong M. Parker K.L. Ezzat S. The transcription activator steroidogenic factor-1 is preferentially expressed in human gonadotroph.J Clin Endocrinol Metab. 1996; 81: 2165-2170Crossref PubMed Scopus (112) Google Scholar showed preferential SF-1 expression in human anterior pituitary gonadotropes, andRamayya et al., 1997Ramayya M.S. Zhou J. Kino T. Segars J. Bondy C.A. Chrousos G.P. Steroidgenic Factor 1 messenger ribonucleic acid expression in steroidogenic and nonsteroidogenic human tissues: Northern blot and in situ hybridization studies.J Clin Endocrinol Metab. 1997; 82: 1799-1806Crossref PubMed Scopus (83) Google Scholar reported the presence of SF-1 mRNA transcripts by northern blot analysis in the spleen and widespread expression throughout many components of the human brain and central nervous system. Similarly, DAX-1 has also been detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in nonsteroidogenic tissues, including human adult and fetal brain, pituitary, and hypothalamus, in addition to its prevalent expression in steroidogenic tissues (Guo et al., 1995Guo W. Burris T.P. McCabe E.R.B. Expression of DAX-1, the gene responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism, in the hypothalamic-pituitary-adrenal/gonadal axis.Biochem Mol Med. 1995; 56: 8-13https://doi.org/10.1006/bmme.1995.1049Crossref PubMed Scopus (149) Google Scholar). Human skin is a target tissue for the steroid hormones released from and tightly controlled by the hypothalamic-pituitary-adrenal-gonadal axis. The capacity for skin to intracellularly metabolize weak circulating androgens such as dehydroepiandrostenedione (DHEA) and androstenedione into the more potent forms, testosterone and dihydrotestosterone (DHT), has been well characterized and these hormones are known to be involved in the regulation of human hair growth. In contrast, it has not yet been proved that human skin is an organ that actively participates in de novo steroid biosynthesis from cholesterol, even though most of the essential components required for androgen and estrogen synthesis, such as cholesterol and the steroidogenic enzymes, namely CYP11A1, CYP17, CYP19, 3β-HSD, 17β-HSD, and 5α-reductase, have been identified in human skin (Courchay et al., 1996Courchay G. Boyera N. Bernard B.A. Mahe Y. Messenger RNA expression of steroidogenesis enzyme subtypes in the human pilosebaceous unit.Skin Pharmacol. 1996; 3: 169-176Crossref Scopus (53) Google Scholar;Slominski et al., 1996Slominski A. Ermak G. Mihm M. ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin.J Clin Endocrinol Metab. 1996; 81: 2746-2749Crossref PubMed Scopus (186) Google Scholar;Venencie et al., 1999Venencie P.Y. Meduri G. Pissard S. Jolivet A. Loosefelt H. Milgrom E. Misrahi M. Luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures.Br J Dermatol. 1999; 141: 438-446https://doi.org/10.1046/j.1365-2133.1999.03036.xCrossref PubMed Scopus (18) Google Scholar). The identification and localization of all the cytochrome P450 steroid hydroxylases in human skin prompted us to investigate whether their expression in skin is controlled in a similar manner as found in the adrenal cortex. In this study, we have shown for the first time using immuno-detection, reverse transcription and polymerase chain reaction (RT-PCR) techniques that DAX-1 and SF-1 appear to be expressed in human skin and its appendages. Our findings also show some differences in expression, in particular, DAX-1 immunoreactivity is predominantly localized to basal cells in the epidermis, whereas SF-1 immunoreactivity is found in all layers except the stratum corneum. Primary human skin keratinocytes were cultured on an irradiated mouse 3T3 feeder layer as previously described (Navsaria et al., 1994Navsaria H.A. Sexton C. Bouvard V. Leigh I.M. Growth of keratinocytes with a 3T3 feeder layer: basic techniques.in: IM Leigh FM Watt Keratinocyte Methods. Cambridge University Press, Cambridge1994: 5-12Google Scholar) in medium consisting of a mixture of Dulbecco's modified Eagle's medium and Ham's F12 in a ratio of 3:1 (vol/vol), supplemented with 10% (vol/vol) fetal calf serum, 0.4 µg hydrocortisone per ml, 10−10 M cholera toxin, 10 ng epidermal growth factor per ml, and 5 µg insulin per ml. HaCaT cells, a spontaneously immortalized keratinocyte cell line (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3270) Google Scholar), were cultured using the same medium as described above but in the absence of a 3T3 feeder layer. The primary keratinocytes and HaCaT cells were cultured in a humidified atmosphere at 37°C with 10% CO2. Primary human preadipocytes were cultured in medium consisting of a mixture of Dulbecco's modified Eagle's medium and Ham's F12 in a ratio of 1:1 (vol/vol), supplemented with 10% (vol/vol) fetal calf serum and 4% (vol/vol) glutamine. αT3-1 cells, a mouse gonadotrope-derived cell line (Windle et al., 1990Windle J.J. Weiner R.I. Mellon P.L. Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice.Mol Endocrinol. 1990; 4: 597-603Crossref PubMed Scopus (437) Google Scholar), were maintained in a monolayer culture in high glucose (4500 µg per ml) Dulbecco's modified Eagle's medium with 10% (vol/vol) fetal calf serum, 100 units penicillin per ml, 100 µg streptomycin per ml, and 125 µg fungizone per ml (Life Technologies, Paisley, U.K.). NCI-H259R human adrenocortical cells (Bird et al., 1993Bird I.M. Hanley N.A. Word R.A. Mathis J.M. McCarthy J.L. Mason J.I. Rainey W.E. Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin II responsive aldosterone secretion.Endocrinology. 1993; 133: 1555-1561Crossref PubMed Scopus (109) Google Scholar) were cultured in a 1:1 (vol/vol) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 15 mM HEPES, 6.25 µg insulin per ml, 6.25 µg transferrin per ml, 6.25 ng selenium per ml, 1.25 mg bovine serum albumin per ml, 5.35 µg linoleic acid per ml, and 2% Ultroser G (Life Technologies, Paisley, U.K.). Pre-adipocytes, αT3-1 cells, and H295R cells were grown at 37°C in a humidified atmosphere with 5% CO2. Facelift skin was obtained from male patients undergoing surgery and was immediately snap-frozen in liquid nitrogen and stored at -80°C until required for hair follicle isolation, sectioning, and protein and RNA extraction. Approval by the East London and City Health Authority Research Ethics Committee (number: T/98/008) for the use of redundant human skin was given. Hair follicles were isolated from facelift skin and transiently maintained in culture as previously described (Philpott et al., 1990Philpott M.P. Green M.R. Kealey T. Human hair growth in vitro.J Cell Sci. 1990; 97: 463-471PubMed Google Scholar). Removal of dermal papilla cells from the isolated hair follicle was carried out according to the method ofJahoda et al., 1984Jahoda C.A.B. Horne K.A. Oliver R.F. Induction of hair growth by implantation of cultured dermal papilla cells.Nature. 1984; 311: 560-561Crossref PubMed Scopus (466) Google Scholar. Briefly, the follicle bulb was dissected away from the isolated hair follicles by means of a scalpel. Using a sterile syringe needle the dermal papilla cells were microdissected out and seeded into T25 tissue culture flasks containing Williams E media (Sigma, Poole, U.K.), supplemented with 15% (vol/vol) fetal calf serum, 100 ng hydrocortisone per ml, 100 units penicillin G per ml (Sigma), 100 µg streptomycin per ml, and 2 mM glutamine. The cells were cultured in a humidified atmosphere at 37°C with 5% CO2 until they reached 70% confluence, after which they were split one in five. Passage two cells were harvested and used for immunocytochemistry and protein extraction. Total RNA was isolated from snap-frozen facelift skin using TRIzol LS Reagent (Gibco BRL, Life Technologies) and from cell cultures using the Qiagen RNeasy Mini kit (Qiagen, Crawley, U.K.). Methods were followed according to the manufacturers' instructions. RNA extracted using either method was treated with DNase I (Gibco BRL, Life Technologies), followed by ethanol precipitation to obtain concentrated RNA. After resuspension in RNase-free water the DNase I was removed using the cleanup protocol from the RNeasy mini kit. The concentration of RNA was estimated by measuring the absorbance at 260 nm and stored at -70°C until required. First-strand DNA synthesis was carried out using a First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Briefly, 5 µg of total RNA in 20 µl of RNase-free water was heated to 65°C for 10 min and chilled on ice, after which it was added to 11 µl of Bulk First-Strand Reaction Mix, 1 µl of a 5 µg per ml Not I-d(T)18 bifunctional primer solution and 1 µl of a 200 mM dithiothreitol solution. The total reaction mix was incubated at 37°C for 1 h and stored at -20°C until use. For PCR, 5 µl of First-strand cDNA were directly pipetted into a mastermix composed of 5 µl of 10 × PCR buffer (Amersham Pharmacia Biotech), 2.5 units of Taq DNA polymerase (Amersham Pharmacia Biotech), 20 mM nucleotide mix (Amersham Pharmacia Biotech), and 40 pmol of primers made up to a total volume of 50 µl using water. PCR of the SF-1 product also required the addition of 5% (vol/vol) dimethyl sulfoxide to the mastermix to remove any secondary structures present in the cDNA caused by GC-rich regions. All primers used were gene-specific and intron spanning and included SF-1 forward 5′-CCTCATCCGGTG TGAGAGC-′3 (nucleotide 987, GenBank accession: D84206), and SF-1 reverse 5′-GGTGCACGTGTAGTGCTTGT-′3 (nucleotide 371, GenBank accession: D84207), which amplify a product of 198 bp, DAX-1 forward 5′-AAGGAGTACGCCTACCTCAA-′3, and DAX-1 reverse 5′-TCCATGCTGACTGTGCCGAT-′3 (nucleotides 1359–1592, GenBank accession: S74720), which amplify a product of 251 bp. Prior to amplification, the PCR reactions were heated for 3 min at 94°C to remove any secondary structures present in the cDNA-RNA heteroduplex. Amplification was carried out for 35 cycles, with denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s in a Perkin-Elmer Thermocycler. PCR products were separated by electrophoresis on a 1.6% agarose gel accompanied by a ϕX174 DNA Hae III digest molecular weight marker (Promega, Hants, U.K.) to determine the size of products, and visualized with ethidium bromide. The PCR products were purified using the QIAquick gel extraction kit (Qiagen, Crawley, U.K.) and sequenced on both strands using the ABI Prism BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Warrington, U.K.). The human adrenocortical H295R cell line was used as a positive control for RT-PCR as they have previously been shown to express DAX-1 by immunofluorescence (Lalli et al., 2000Lalli E. Ohe K. Hindelang C. Sassone-Corsi P. Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA.Mol Cell Biol. 2000; 20: 4910-4921Crossref PubMed Scopus (104) Google Scholar) and SF-1 by northern analysis (Leers-Sucheta et al., 1997Leers-Sucheta S. Morohashi K. Mason J.I. Melneri M.H. Synergistic activation of the human type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase promoter by the transcription factor Steroidogenic Factor-1/Adrenal 4-binding Protein and phorbol ester.J Biol Chem. 1997; 272: 7960-7967Crossref PubMed Scopus (154) Google Scholar). DNA sequencing of products amplified from skin and H295R cDNA confirmed their identity with SF-1 and DAX-1 on the GenBank database. Six-micrometer thick longitudinal sections were cut from hair-bearing human male scalp skin and mounted on superfrost microscope slides. The slides were stored at -80°C until needed for immunohistochemistry. The slides were left to air-dry at room temperature for 20 min followed by treatment in 3% (vol/vol) H2O2 in methanol for 15 min to block endogenous peroxidase activity. The sections were preincubated with neat horse serum for 20 min and then incubated with a rabbit polyclonal antibody raised against mouse SF-1 (Upstate Biotechnology, Lake Placid, NY) at a concentration of 6.0 µg per ml, and a rabbit polyclonal antibody raised against human DAX-1 (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 0.5 µg per ml, for 1 h at room temperature in antibody diluent (5 mM Tris, 140 mM NaCl, 6 mM NaN3, 16 mM HCl, 0.4 mg per ml bovine serum albumin, and 5.6 µg per ml caesin, pH 7.6). Prior to use of the primary antibodies a titration study was carried out to determine the optimum concentration for their use on histological sections. Secondary and tertiary complexes were formed using the Vectastain Universal Elite ABC kit (Vector Laboratories, Burlingame, CA). Briefly, specimens were washed in Tris-buffered saline (TBS) 20 mM Tris-base; pH 7.6, 140 mM NaCl and incubated with an antihorse biotinylated IgG in 6% blocking serum (Vector Laboratories) for 45 min at room temperature. The sections were subsequently washed in TBS followed by incubation with an avidin-biotin-peroxidase complex (Vector Laboratories) for 45 min at room temperature. Immunoreactivity was visualized using diaminobenzidine (DAB) (BioGenex, San Ramon, CA) as a chromogenic substrate in H2O2. Sections were dehydrated and then mounted in DePex mounting medium (BDH Laboratory Supplies, Poole, U.K.). Omission of the primary antibody for negative controls resulted in no detection demonstrating specificity of the primary antibodies (data not shown). Dermal papilla cells, HaCaT cells, normal human keratinocytes, preadipocytes, and αT3-1 cells were trypsinized and seeded at a density of 1 × 105 cells on to flame-sterilized glass coverslips that had been placed in 6-well tissue culture dishes. The cells were grown in their respective medium (as described above) for 2d before immunocytochemistry was carried out. After 48 h, culture media was aspirated and the coverslips were washed three times with TBS. Cell fixation and blocking of endogenous peroxidases was carried out by incubating the coverslips with a 3% (vol/vol) H2O2 in methanol solution for 15 min at room temperature. The cells were washed three times in TBS and treated with TBS/0.1% (vol/vol) Triton X-100 for 15 min at room temperature to permeabilize the cells. This was followed by three washes in TBS, incubation for 20 min at room temperature in neat horse serum to block nonspecific binding, and incubation for 1 h at room temperature in either 0.5 µg per ml anti-DAX-1 antibody or 12.0 µg per ml anti-SF-1 antibody in antibody diluent. After incubation in primary antibody was completed, the cells were washed three times in TBS, followed by secondary and tertiary complexing using the Vectastain Universal Elite ABC kit and color development using DAB, as described above. Control experiments included omission of the primary antibody (negative control), which resulted in no detection, demonstrating specificity of the primary antibodies, and by using αT3-1 cells as a positive control for DAX-1 and SF-1 immunoreactivity, which were previously shown to express SF-1 by northern blotting (Barnhart and Mellon, 1994Barnhart K.M. Mellon P.L. The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone α-subunit gene in pituitary gonadotropes.Mol Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (203) Google Scholar), and DAX-1 by RNase protection assay (Ikeda et al., 1996Ikeda Y. Swain A. Weber T.J. et al.Steroidogenic factor 1 and DAX-1 co-localize in multiple cell lineages: potential links in endocrine development.Mol Endocrinol. 1996; 10: 1261-1272Crossref PubMed Scopus (267) Google Scholar). Protein was extracted from 5 × 106 cells of each cell type grown in T25 flasks using a modified method ofSchreiber et al., 1989Schreiber E. Matthias P. Müller M.M. Schaffner W. Rapid detection of octomer binding proteins with “mini-extracts”, prepared from a small number of cells.Nucl Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3880) Google Scholar. Briefly, culture medium was removed and the cells were washed in ice-cold phosphate-buffered saline (PBS). The cells were then harvested by scraping in cold PBS and pelleted by centrifugation at 1300 × g, 4°C, for 5 min. The supernatant was discarded and the cells were resuspended in 400 µl of cold buffer A (10 mM HEPES pH 7.9; 10 mM KCl, 0.1 mM EDTA; 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM PMSF). The cells were left on ice for 15 min, followed by the addition of 25 µl of a 10% Nonidet (NP-40) solution made in buffer A. The cells were immediately vortexed for a duration of 10 s and then centrifuged at 16000 × g for 30 s. The supernatant was removed prior to the resuspension of the pellet in 50 µl of buffer B (20 mM HEPES pH 7.9; 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA; 1 mM dithiothreitol; 1 mM phenylmethylsulfonyl fluoride) and then the cells were thoroughly mixed on ice for 15 min using a shaking platform. The nuclear extract was centrifuged for 5 min, 16000 × g, at 4°C and the supernatant containing the nuclear protein was collected and stored at -70°C. Nuclear protein concentration was determined using the Bradford assay (Bio-Rad, Hemel Hempstead, U.K.) with bovine serum albumin as standard. Fifteen micrograms of each nuclear protein extract was mixed with a 2:1 volume of sample buffer [125 mM Tris (hydroxymethyl) aminomethane pH 6.8, 5% (vol/vol) β-mercaptoethanol, 20% (vol/vol) glycerol, 4% sodium dodecyl sulfate, and 0.02% bromophenol blue] and separated by SDS-polyacrylamide gel electrophoresis, using precast 10% Tris-HCl polyacrylamide gels (Bio-Rad, Hemel Hempstead, U.K.) for 1 h at 90 V; thereafter, proteins were electrotransferred to a nitrocellulose membrane for 1 h at 90 V. The membrane was blocked using 5% (vol/vol) non-fat dry milk in TBS/0.1% (vol/vol) Tween-20, pH 7.5, for 1.5 h at 20°C, prior to overnight incubation in 0.08 µg per ml anti-DAX-1 in blocking buffer, at 4°C. The nitrocellulose membrane was washed three times with TBS/0.1% (vol/vol) Tween-20 for 5 min each, and subsequently incubated with goat anti-rabbit horseradish peroxidase (DAKO, Cambridge, U.K.) at 0.03 µg per ml in blocking buffer for 1.5 h at room temperature. Following three washes in TBS/0.1% (vol/vol) Tween-20, the protein bands were visualized using an enhanced chemiluminescence ECL detection system (Amersham Pharmacia Biotech). Immunohistochemistry on human facelift skin sections for DAX-1 and SF-1 revealed distinctive differential nuclear staining for both transcription factors in specific regions of the human hair follicle and skin Figure 1. Within the epidermis, prominent nuclear and cytoplasmic DAX-1 expression was detected in the basal layer, with decreasing expression towards the differentiated suprabasal keratinocytes Figure 1a, whereas nuclear SF-1 immunoreactivity was diffuse across all strata of the epidermis, namely the stratum basale, stratum spinosum, and stratum granulosum Figure 1b. Immunolocalization for both transcription factors was also apparent in the dermal fibroblasts Figure 1a, b. In the human hair follicle, there was a distinguishable difference in DAX-1 and SF-1 staining Figure 1c, d. Strong nuclear staining for DAX-1 was confined to the outer root sheath with weaker expression in the inner root sheath, matrix cells Figure 1c and significantly lower immunoreactivity in the dermal papilla cells Figure 1c, e. There was no difference in distribution for SF-1 throughout the hair follicle in comparison with DAX-1 expression, but the levels of immunoreactivity for SF-1 in the outer root sheath, inner root sheath, matrix cells Figure 1d, and dermal papilla cells of the hair bulb Figure 1d, f were stronger than that for DAX-1. A similar pattern of expression for both orphan nuclear receptors was detected in the exocrine gland" @default.
• W2042180438 created "2016-06-24" @default.
• W2042180438 creator A5022276400 @default.
• W2042180438 creator A5062866219 @default.
• W2042180438 creator A5090324778 @default.
• W2042180438 date "2001-12-01" @default.
• W2042180438 modified "2023-10-12" @default.
• W2042180438 title "Transcriptional Regulators of Steroidogenesis, DAX-1 and SF-1, are Expressed in Human Skin" @default.
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