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Enhancing 1α-Hydroxylase Activity with the 25-Hydroxyvitamin D-1α-Hydroxylase Gene in Cultured Human Keratinocytes and Mouse Skin

2001; Elsevier BV; Volume: 116; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.2001.01360.x

1523-1747

Tai C. Chen, Xue Hong Zhu, Michael T. Holick, Xiangfu Kong, Michael F. Holick, John N. Flanagan, Lyman W. Whitlatch,

Dermatology and Skin Diseases

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) and its analogs are used to treat psoriasis because of their potent antiproliferative activity. They have the potential for causing hypercalcemia, however, and patients often become resistant to the drug. We examined the feasibility of enhancing the cutaneous production of 1α,25(OH)2D3 using a human 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase) plasmid. The 1α-OHase gene was fused to the green fluorescent protein gene (1α-OHase-GFP) driven by the cytomegalovirus promoter. Transfection of cultured normal human keratinocytes with the 1α-OHase-GFP plasmid resulted in a marked increase in the expression of 1α-OHase-GFP in the mitochondria. Transfection of keratinocytes with 1α-OHase-GFP or 1α-OHase plasmids in vitro enhanced the 1α-OHase activity substantially and increased the sensitivity of the keratinocytes to the antiproliferative effect of 25(OH)D3. The 1α-OHase-GFP plasmid was topically applied to shaved C57/BL6 mice. Twenty-four hours after topical application, immunohistochemical analysis of the skin for 1α-OHase-GFP revealed the presence of 1α-OHase-GFP in the epidermis and epidermal appendages including the hair follicles. The results from this study offer a unique new approach for the topical treatment of hyperproliferative disorders such as psoriasis and skin cancer using the 1α-OHase gene that could locally increase the production of 1α,25(OH)2D3 without causing hypercalcemia or resistance. 1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) and its analogs are used to treat psoriasis because of their potent antiproliferative activity. They have the potential for causing hypercalcemia, however, and patients often become resistant to the drug. We examined the feasibility of enhancing the cutaneous production of 1α,25(OH)2D3 using a human 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase) plasmid. The 1α-OHase gene was fused to the green fluorescent protein gene (1α-OHase-GFP) driven by the cytomegalovirus promoter. Transfection of cultured normal human keratinocytes with the 1α-OHase-GFP plasmid resulted in a marked increase in the expression of 1α-OHase-GFP in the mitochondria. Transfection of keratinocytes with 1α-OHase-GFP or 1α-OHase plasmids in vitro enhanced the 1α-OHase activity substantially and increased the sensitivity of the keratinocytes to the antiproliferative effect of 25(OH)D3. The 1α-OHase-GFP plasmid was topically applied to shaved C57/BL6 mice. Twenty-four hours after topical application, immunohistochemical analysis of the skin for 1α-OHase-GFP revealed the presence of 1α-OHase-GFP in the epidermis and epidermal appendages including the hair follicles. The results from this study offer a unique new approach for the topical treatment of hyperproliferative disorders such as psoriasis and skin cancer using the 1α-OHase gene that could locally increase the production of 1α,25(OH)2D3 without causing hypercalcemia or resistance. 1α,25-dihydroxyvitamin D3 25-hydroxyvitamin D-1α-hydroxylase 25-hydroxyvitamin D-1α-hydroxylase-green fluorescent protein. Vitamin D is biologically inert and requires two successive hydroxylations first in the liver to form 25-hydroxyvitamin D3 (25(OH)D3) and then in the kidney to its biologically active form 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) (Holick and Favus, 1999Holick M.F. Favus M.J. Primer.in: on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 4th edn. Lippincott-Raven, Philadelphia1999: 92-98Google Scholar). Once formed, 1α,25(OH)2D3 goes to its target tissues and interacts with a specific nuclear receptor, the vitamin D receptor (Stumpf et al., 1979Stumpf W.E. Sar M. Reid F.A. Tanaka Y. DeLuca H.F. Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, stomach, kidney, skin, pituitary, and parathyroid.Science. 1979; 206: 1188-1190Crossref PubMed Scopus (561) Google Scholar). 1α,25(OH)2D3 is not only important in calcium metabolism, but also has pro-differentiation and cell growth inhibitory activities (Tanaka et al., 1982Tanaka H. Abe E. Miyaura C. Kuribayashi T. Konno K. Nishii Y. Suda T. 1,25-Dihydroxycholecalciferol and human myeloid leukemia cell line (HL-60): the presence of cytosol receptor and induction of differentiation.Biochem J. 1982; 204: 713-719Crossref PubMed Scopus (380) Google Scholar;Hosomi et al., 1983Hosomi J. Hosoi J. Abe E. Suda T. Kuroki T. Regulation of terminal differentiation of cultured mouse epidermal cells by 1α,25-dihydroxyvitamin D3.Endocrinology. 1983; 113: 1950-1957Crossref PubMed Scopus (428) Google Scholar;Smith et al., 1986Smith E.L. Walworth N.D. Holick M.F. Effect of 1,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions.J Invest Dermatol. 1986; 86: 709-714Crossref PubMed Scopus (420) Google Scholar;Chen et al., 1993Chen T.C. Person K. Uskokovic M.R. Horst R.L. Holick M.F. An evaluation of 1,25-dihydroxyvitamin D3 analogues on the proliferation and differentiation of cultured human keratinocytes, calcium metabolism, and the differentiation of human HL-60 cells.J Nutr Biochem. 1993; 4: 49-57Abstract Full Text PDF Scopus (56) Google Scholar). The enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-OHase) that is responsible for producing 1α,25(OH)2D3 is a cytochrome P450 enzyme that is present in the mitochondria. This enzyme is not only present in the kidney, but is found in many other tissues including activated macrophage, prostate, and skin (Bikle et al., 1986Bikle D.D. Nemanic M.D. Whitney J.O. Elias P.O. Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3.Biochem. 1986; 25: 1545-1548Crossref PubMed Scopus (167) Google Scholar;Adams et al., 1990Adams J.S. Beeker T.G. Hongo T. Clemens T.L. Constitutive expression of a vitamin D 1-hydroxylase in a myelomonocytic cell line: a model for studying 1,25-dihydroxyvitamin D production in vitro.J Bone Min Res. 1990; 5: 1265-1269Crossref PubMed Scopus (31) Google Scholar;Schwartz et al., 1998Schwartz G.G. Whitlatch L.W. Chen T.C. Lokeshwar B.L. Holick M.F. Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3.Cancer Epidemiol, Biomark Prev. 1998; 7: 391-395PubMed Google Scholar). The cDNAs encoding the mouse, rat, and human 1α-OHase have been cloned (Fu et al., 1997Fu G.K. Lin D. Zhang M.Y. Bikle D.D. Shackleton C.H. Miller W.L. Portale A.A. Cloning of human 25-hydroxyvitamin D-1α-hydroxylase and mutations causing vitamin D-dependent rickets type 1.Molecul Endocrinol. 1997; 11: 1961-1970Crossref PubMed Google Scholar;Takeyama et al., 1997Takeyama K.I. Kitanaka S. Sato T. Kobori M. Yanagisawa J. Kato S. 25-Hydroxyvitamin D3 1α-hydroxylase and vitamin D synthesis.Science. 1997; 277: 1827-1830https://doi.org/10.1126/science.277.5333.1827Crossref PubMed Scopus (421) Google Scholar;Kitanaka et al., 1998Kitanaka S. Takeyama K. Murayama A. et al.Inactivating mutations in the human 25-hydroxyvitamin D3 1α-hydroxylase gene in patients with pseudovitamin D-deficient rickets.N Engl J Med. 1998; 338: 653-661Crossref PubMed Scopus (275) Google Scholar;Kong et al., 1999Kong X.F. Zhu X.H. Pei Y.L. Jackson D.M. Holick M.F. Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1α- hydroxylase gene.Proc Natl Acad Sci. 1999; 96: 6988-6993Crossref PubMed Scopus (100) Google Scholar). The human renal and nonrenal 1α-OHase cDNA sequences are 100% identical. The presence of 1α-OHase in keratinocytes suggests an autocrine/paracrine role for 1α,25(OH)2D3 that may be responsible for locally modulating cell proliferation and differentiation. The potent antiproliferative and pro-differentiating effects of 1α,25(OH)2D3 and its analogs on keratinocytes has led to the development of these compounds as new therapies for the hyperproliferative skin disease psoriasis (Morimoto et al., 1986Morimoto S. Onishi T. Imanaka S. et al.Topical administration of 1,25-dihydroxyvitamin D3 for psoriasis: report of five cases.Calcif Tissue Int. 1986; 38: 119-122Crossref PubMed Scopus (66) Google Scholar;Kragballe et al., 1988Kragballe K. Beck H.I. Sogaard H. Improvement of psoriasis by a topical vitamin D3 analogue (MC903) in a double-blind study.Br J Dermatol. 1988; 119: 223-230Crossref PubMed Scopus (180) Google Scholar;Smith et al., 1988Smith E.L. Pincus S.H. Donovan L. Holick M.F. A novel approach for the evaluation and treatment of psoriasis: oral or topical use of 1,25-dihydroxyvitamin D3 can be a safe and effective therapy for psoriasis.J Am Acad Dermatol. 1988; 19: 516-528Abstract Full Text PDF PubMed Scopus (177) Google Scholar;Van De Kerkhof et al., 1989Van De Kerkhof P. Van Bokhoven M. Zultak M. Czarnetzki B. A double-blind study of topical 1α,25-dihydroxyvitamin D3 in psoriasis.Br J Dermatol. 1989; 120: 661-664Crossref PubMed Scopus (39) Google Scholar;Perez et al., 1996Perez A. Chen T.C. Turner A. Raab R. Bhawan J. Poche P. Holick M.F. Efficacy and safety of topical calcitriol (1,25-dihydroxyvitamin D3) for the treatment of psoriasis.Br J Dermatol. 1996; 134: 238-246Crossref PubMed Scopus (72) Google Scholar). Topically applied active vitamin D drugs, however, can be absorbed through the skin to cause hypercalcemia (Bower et al., 1991Bower M. Colston K.W. Stein R.C. Hedley A. Gazet J.C. Ford H.T. Combes R.C. Topical calcipotriol treatment in advanced breast cancer.Lancet. 1991; 337: 701-702Abstract PubMed Scopus (113) Google Scholar;Hoech et al., 1994Hoech H.C. Laurberg G. Laurberg P. Hypercalacemia crisis after excessive topical use of a vitamin D derivative.J Intern Med. 1994; 235: 281-282Crossref PubMed Scopus (30) Google Scholar). Furthermore only 40%-60% of patients have a good response to this therapy and a resistance to the therapy may be developed after months of use. Therefore there is a need for an alternative strategy to increase the local production of 1α,25(OH)2D3 in hyperproliferative cells. During the past decade, advances in the introduction of genes to tissues and organs have greatly enhanced the prospect of gene therapy for humans (Katz, 1999Katz S.I. Thematic Review Series VI: Skin gene therapy.Proc Assoc Am Physicians. 1999; 111: 183-219Crossref PubMed Scopus (1) Google Scholar). The topical application of the 1α-OHase gene to the skin to increase local 1α-OHase activity may provide a unique and effective therapeutic approach for hyperproliferative disorders of the skin such as psoriasis. We report on the feasibility of introducing the 1α-OHase gene into cultured normal human keratinocytes, which increased the 1α-OHase activity and sensitivity to the antiproliferative activity of 25(OH)D3in vitro. We also show that the topical application of the 1α-OHase cDNA plasmid on mice enhanced the cutaneous expression of the 1α-OHase in vivo. Polymerase chain reaction (PCR) mutagenesis was used to generate the 1α-OHase-green fluorescent protein (1α-OHase-GFP) fusion construct from 1α-OHase cDNA (Kong et al., 1999Kong X.F. Zhu X.H. Pei Y.L. Jackson D.M. Holick M.F. Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1α- hydroxylase gene.Proc Natl Acad Sci. 1999; 96: 6988-6993Crossref PubMed Scopus (100) Google Scholar). The forward oligonucleotide primer (5′-GCA CGA ATT CAA TGA CCC AGA C-3′) was designed to introduce an EcoRI endonuclease site 10 base pairs upstream of the 1α-OHase start codon. The reverse oligonucleotide primer (5′-GAC CGT AAG CTT CTA TCT GTC-3′) was designed to mutate the endogenous stop codon and to introduce a Hind III endonuclease site just downstream of the stop codon. The resulting 1α-OHase(-stop) fragment was ligated into pCR3.1 T/A cloning vector (Invitrogen, Carlsbad, CA). Multiple positive clones were isolated and confirmed by restriction analysis and nucleotide sequencing. The 1α-OHase(-stop).pCR3.1 positive clone was ligated to pEGFP vector (Clontech, Palo Alto, CA). 1α-OHase(-stop).pEGFP plasmid DNA was purified using Qiagen Maxi Purification Kit (Qiagen, Chatsworth, CA). Normal human keratinocytes were obtained from neonatal foreskin as previously described (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-334Abstract Full Text PDF PubMed Scopus (3722) Google Scholar) and grown in serum-free defined medium (Chen et al., 1995Chen T.C. Persons K. Liu W.W. Chen M.L. Holick M.F. The antiproliferative and differentiative activities of 1,25-dihydroxyvitamin D3 are potentiated by epidermal growth factor and attenuated by insulin in cultured human keratinocytes.J Invest Dermatol. 1995; 104: 113-117Crossref PubMed Scopus (58) Google Scholar). Transfection of keratinocytes was carried out using LipofectAmine Kit (Life Technologies, Grand Island, NY). Cells were grown to about 60% confluency in the absence of antibiotics, at which time the cells were exposed to fresh medium and added with freshly prepared 1α-OHase-GFP cDNA-LipofectAmine Reagent complexes (0.05 ml) as described in the protocol supplied by the company. The cells were incubated for 3 h at 37°C with 5% CO2 and then the transfection solution was removed, fresh medium was added, and the cells were incubated for an additional 18–48 h. Transfection efficiency of 1α-OHase-GFP in normal human keratinocytes between different experiments ranged from 20% to 30%. The expression and the appearance of 1α-OHase-GFP fusion protein inside the transfected keratinocytes were confirmed by detecting the green fluorescence using scanning laser confocal microscopy (InSight Plus, Meridian Instruments, Okemos, MI) at 600×. The normal human keratinocyte transfection conditions were the same as for confocal microscopy. Twenty-four hours post-transfection, cells were washed once with 1 × phosphate-buffered saline (PBS), trypsinized for 10 min in 1 × trypsin ethylenediamine tetraacetic acid (EDTA), and spun for 10 min at 5000 rpm. Total RNA isolation was performed using SV Total RNA Isolation System (Promega, Madison, WI). Two micrograms of total RNA isolated from treated cells were reverse transcribed into single stranded cDNA at 37°C for 2 h using Superscript II RNase H Reverse Transcriptase (Life Technologies, Rockville, MD). Two hundred nanograms of single stranded cDNA template were used in each PCR. The following sequence specific primers were designed: GFP sense primer 5′-CAA GCA GAA GAA CGG CAT CAA-3′ and antisense primer 5′-GGA CTG GGT GCT CAG GTA GTG-3′; 1α-OHase-GFP sense primer 5′-GAG GTG CAG CCT GAG CCA-3′ and antisense primer 5′-CCA GCT CGA CCA GGA GGT-3′. The PCR protocol was as follows: 95°C for 10 min and then for 40 cycles, denaturation at 95°C for 15 s, and anneal/extend at 60°C for 1 min. One-fifth of the product was analyzed on a 4% agarose gel. Twenty-four hours post-transfection, cell lysates from normal human keratinocytes transfected with either GFP alone or 1α-OHase-GFP were prepared as follows: cells were washed once with ice-cold PBS, scraped with a rubber policeman in ice-cold RIPA buffer [1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 5 mM EDTA, and 10 mM sodium phosphate, pH 7.2, with complete protease inhibitor mixture], and sonicated at a concentration of 1 × 106 cells per 100 µl. Approximately 10 µg of protein in total lysate were loaded per lane on an 8% SDS polyacrylamide gel. The gel was electrophoresed and transferred onto a nitrocellulose membrane. The membrane was then rinsed briefly with 5% milk-TBST blocking buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 30 min with gentle shaking. The membrane was further incubated with anti-GFP antibody (Clontech) in 0.5% milk-TBST for 1 h with gentle shaking. It was then washed three times for 10 min each with 0.5% milk-TBST followed by an antirabbit peroxidase conjugate for 30 min in 0.5% milk-TBST. It was washed twice for 10 min each in TBST and twice for 10 min each in TBS and chemiluminescent substrate luminal/iodophenol (New England Nuclear, Boston, MA) was added as per the manufacturer's instruction. The membrane was exposed to X-ray film for 3 s to 5 min. The 1α-OHase enzyme activity was determined 24 h after transfection in the presence of 0.1 μCi of 3H-25(OH)D3 (New England Nuclear) and 10 μM 1,2-dianilinoethane by high performance liquid chromatography using methylene chloride:isopropanol (19:1) as the mobile phase as described previously (Schwartz et al., 1998Schwartz G.G. Whitlatch L.W. Chen T.C. Lokeshwar B.L. Holick M.F. Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3.Cancer Epidemiol, Biomark Prev. 1998; 7: 391-395PubMed Google Scholar). The retention volumes for 3H-25(OH)D3 and 3H-1α,25(OH)2D3 were confirmed with standard nonradioactive 25(OH)D3 and 1α,25(OH)2D3. The enzyme activity was expressed as pmol of 1α,25(OH)2D3 per mg protein per h. The antiproliferative effects of 25(OH)D3 on transfected normal human cultured keratinocytes were investigated in 24-well plates by determining 3H-thymidine incorporation. Twenty-four hours after transfection, keratinocytes were treated with 25(OH)D3 (10-9, 10-8, 10-7 M) for 18 h. Forty-two hours after transfection, the dosing medium was replaced with 0.5 ml of fresh basal medium containing [methyl-3H]thymidine (New England Nuclear) and 3H-thymidine incorporation into DNA was performed as described previously (Smith et al., 1986Smith E.L. Walworth N.D. Holick M.F. Effect of 1,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions.J Invest Dermatol. 1986; 86: 709-714Crossref PubMed Scopus (420) Google Scholar). The results were expressed as percentage of radioactivity incorporated in the absence of 25(OH)D3. Eight-week-old C57/BL6 female mice in telogen phase were depilated with a wax/rosin mixture on their dorsal surface as described previously (Schilli et al., 1997Schilli M.B. Ray S. Paus R. Obi-Tabot E. Holick M.F. Control of hair growth with parathyroid hormone (7-34).J Invest Dermatol. 1997; 108: 928-932Crossref PubMed Scopus (43) Google Scholar). Depilation was followed by tape-stripping the stratum corneum twice with Hair Remover Wax Strip Kit (Del Laboratories, Farmingdale, NY). The skin surface was then cleaned with ethanol and allowed to cool for 15 min. Twenty microliters of a 1 µg per µl solution of naked plasmid DNA of 1α-OHase-GFP plasmid, or water alone, were applied to a 1 cm2 area of the pretreated skin as previously described (Fan et al., 1999Fan H. Lin Q. Morrissey G.R. Khavari P.A. Immunization via hair follicles by topical application of naked DNA to normal skin.Nat Biotechnol. 1999; 17: 870-872https://doi.org/10.1038/12856Crossref PubMed Scopus (164) Google Scholar;Yu et al., 1999Yu W.H. Kashani-Sabet M. Liggitt D. Moore D. Heath T.D. Debs R.J. Topical gene delivery to murine skin.J Invest Dermatol. 1999; 112: 370-375https://doi.org/10.1046/j.1523-1747.1999.00513.xCrossref PubMed Scopus (79) Google Scholar). Twenty-four hours after topically applying naked cDNA to abraded mouse skin, biopsies were taken, embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), and flash frozen, and 5 µm sections were taken. Sections were fixed in 100% acetone and blocked with 4% fatty acid free bovine serum albumin (BSA)/0.1% Triton X-100 in PBS for 1 h at room temperature. The sections were then incubated with 40 µg per ml of rabbit anti-GFP antibody (Clontech) in 4% BSA-PBS solution overnight at 4°C. Following three washes in PBS for 5 min the next day, the sections were incubated with goat antirabbit-biotin label antibody (Vector, Burlingame, CA) at the appropriate dilution for 2 h at room temperature. The sections were washed three times with PBS and incubated with a streptavidin-alkaline phosphatase conjugated antibody (Vector) for 1 h at room temperature at the appropriate dilution. Fluorescence was developed using Phosphatase Substrate kit I (Vector) for approximately 10 min. Sections were then rinsed in distilled water, mounted with Permount (Fisher, Pittsburgh, PA), and examined for fluorescence using a Nikon fluorescence microscope (Melville, NY) with a rhodamine filter system and standard compound light microscope at 200×. The expression vector encoding the 1α-OHase-GFP fusion protein driven by the cytomegalovirus promoter was transfected into normal human cultured keratinocytes to examine the expression and the appearance of the cellular distribution of the 1α-OHase-GFP fusion protein using scanning laser confocal microscopy. The fluorescence of the 1α-OHase-GFP protein in live transfected keratinocytes showed an organelle distribution that was consistent with the expected location for the cytochrome P450-1α-OHase (Figure 1a) (Jones and Holick, 1999Jones G. Holick M.F. Vitamin D. Molecular Biology, Physiology, and Clinical Applications. Humana Press, Totowa, NJ1999: 57-84Google Scholar). The same live transfected keratinocytes were also stained with a mitochondrial specific dye (Figure 1b). When both images were superimposed the resulting image showed colocalization of the 1α-OHase-GFP green fluorescence with the mitochondrial red fluorescence that appears yellowish-green, confirming that the 1α-OHase-GFP protein was expressed in the mitochondria (Figure 1c). This is in contrast to a live keratinocyte that was transfected with the control GFP plasmid. There was uniform fluorescence throughout the cytoplasm, which is consistent with the GFP protein being expressed in the cytoplasm (Figure 1d). To confirm the expression of the 1α-OHase-GFP, we examined the protein from transfected normal human keratinocytes. Normal human keratinocytes transfected with 1α-OHase-GFP cDNA plasmid produced the appropriate 84 kDa size fusion protein on Western blot analysis (Figure 2a); this was not observed in normal human keratinocytes transfected with GFP cDNA plasmid alone. Furthermore, the appropriate 26.3 kDa size GFP product was observed only in normal human keratinocytes transfected with GFP cDNA plasmid and not in normal human keratinocytes transfected with 1α-OHase-GFP cDNA plasmid (Figure 2b). We examined the 1α-OHase-GFP mRNA expression of the normal human keratinocytes transfected with 1α-OHase-GFP cDNA plasmid. By using sequence specific primers for reverse transcriptase PCR to amplify an amplicon that spanned the 1α-OHase-GFP junction, only one PCR product of 175 bp was observed in normal human keratinocytes transfected with 1α-OHase-GFP cDNA plasmid and it was not observed in normal human keratinocytes transfected with GFP cDNA plasmid alone (Figure 2c). Sequence specific primers to GFP alone, however, produced PCR products of 150 bp that were observed in normal human keratinocytes transfected with both GFP cDNA plasmid and 1α-OHase-GFP cDNA plasmid (Figure 2d). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers were also used to confirm equivalent amounts of cDNA (Figure 2e). No PCR products were observed in PCRs containing total RNA of each sample or primers alone, assuring that there was no plasmid or genomic contamination (data not shown).Figure 2The expression of GFP and 1α-OHase-GFP protein and mRNA in normal human keratinocytes transfected with GFP or 1α-OHase-GFP cDNA plasmid. Protein and mRNA expression was observed 24 h after transfection. (a, b) Western blot analysis of lysates from normal human keratinocytes transfected with either GFP (left lane) or 1α-OHase-GFP (right lane) cDNA plasmid. (c -e) Reverse transcriptase PCR products of RNA isolated from normal human keratinocytes transfected with either GFP (left lane) or 1α-OHase-GFP (right lane) cDNA plasmid using sequence specific primers spanning the 1α-OHase-GFP fusion junction (c), sequence specific primers to the GFP epitope tag (d), or sequence specific primers to GAPDH (e).View Large Image Figure ViewerDownload (PPT) To determine whether the expressed 1α-OHase-GFP was functional, we examined the efficiency in the conversion of 3H-25(OH)D3 to 3H-1α,25(OH)2D3 in keratinocytes transfected with 1α-OHase-GFP. A representative elusion profile from high performance liquid chromatography analysis shows an increased conversion of 3H-25(OH)D3 to 3H-1α,25(OH)2D3 in transfected normal human keratinocytes with 1α-OHase-GFP plasmid (Figure 3b) compared with the endogenous level in the GFP transfected control (Figure 3a). High performance liquid chromatography analysis revealed an 85% ± 4.7% (mean ± SEM; n = 6) increase in the conversion of 3H-25(OH)D3 to 3H-1α,25(OH)2D3 in keratinocytes transfected with 1α-OHase-GFP above basal activity (transfected with GFP vector alone; n = 3) (Figure 3c). The 1α-OHase gene alone without the GFP tag was also transfected into keratinocytes. These cells showed a 111% ± 5.7% (mean ± SEM; n = 3) increase in the conversion of 3H-25(OH)D3 to 3H-1α,25(OH)2D3. This was not statistically significantly different from the 1α-OHase-GFP transfected cells demonstrating that the 26.3 kDa GFP tag did not significantly alter the function of 1α-OHase. There was no difference in 1α-OHase activity in cells transfected with either GFP cDNA plasmid, PCR 3.1 vector plasmid, or LipofectAmine alone (data not shown). Once we established that keratinocytes transfected with 1α-OHase and 1α-OHase-GFP had enhanced 1α-OHase activity, we evaluated whether the enhanced conversion of 25(OH)D3 to 1α,25(OH)2D3 would result in an increased sensitivity to the antiproliferative effects of 25(OH)D3. The antiproliferative activity of 25(OH)D3 was determined in the transfected keratinocytes using the 3H-thymidine incorporation assay (Smith et al., 1986Smith E.L. Walworth N.D. Holick M.F. Effect of 1,25-dihydroxyvitamin D3 on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions.J Invest Dermatol. 1986; 86: 709-714Crossref PubMed Scopus (420) Google Scholar;Chen et al., 1995Chen T.C. Persons K. Liu W.W. Chen M.L. Holick M.F. The antiproliferative and differentiative activities of 1,25-dihydroxyvitamin D3 are potentiated by epidermal growth factor and attenuated by insulin in cultured human keratinocytes.J Invest Dermatol. 1995; 104: 113-117Crossref PubMed Scopus (58) Google Scholar). In Figure 4, keratinocytes transfected with 1α-OHase showed significant decreases in 3H-thymidine incorporation of 25 ± 5.0%, 26 ± 3.4%, and 54 ± 0.9% at 10-9 M, 10-8 M, and 10-7 M of 25(OH)D3 (mean ± SEM; n = 8–24), respectively, compared to their respective liposome or vector alone control. There was no significant difference in 25(OH)D3 responses in cells transfected with either LipofectAmine or vector alone (Figure 4). Naked 1α-OHase-GFP plasmid DNA in water was topically applied to mouse skin to see whether the gene could be expressed in vivo in the epidermis. The expression of the 1α-OHase-GFP protein was detected with a GFP antibody. No cross-reactivity of the GFP antibody was detected in either the epidermis or dermis of the skin treated with water vehicle (Figure 5a). As indicated in Figure 5(b) by the red fluorescence of the fluorophore secondary antibody (Vector Bioscience, Eugene, OR), however, there was marked expression of the 1α-OHase-GFP in all the cells in the epidermis of the mouse that received a single topical application of the 1α-OHase-GFP plasmid DNA 24 h previously. These results were confirmed using compound light microscopy with the 1α-OHase-GFP located in the epidermis and epidermal appendages indicated by the red-brown staining observed in the cross-section of skin in Figure 5(d) compared with skin from a control mouse that was treated with water vehicle (Figure 5c). Results from this study demonstrate the feasibility of using the 1α-OHase plasmid for gene therapy to treat hyperproliferative disorders of the skin such as psoriasis and skin cancer. In vitro the increase in mitochondrial expression of 1α-OHase resulted in increased conversion of 25(OH)D3 to 1α,25(OH)2D3. The enhanced 1α-hydroxylase activity imparted increased sensitivity to the antiproliferative activity of 25(OH)D3, presumably because of the increased conversion of 25(OH)D3 to 1α,25(OH)2D3 within the transfected keratinocytes. Thus, topically applying a plasmid construct of the 1α-OHase gene to psoriatic lesions should result in the local enhanced production of 1α,25(OH)2D3 in psoriatic skin cells. The increased intracellular levels of 1α,25(OH)2D3 should in turn restore cells to normal proliferation and differentiation, resulting in remission in the disease similar to if the skin lesion was treated topically with 1α,25(OH)2D3 or one of its analogs. This may be a preferred method of treatment not only for psoriasis but also for other hyperproliferative disorders of the skin such as actinic keratoses and skin cancer. Increasing 1α,25(OH)2D3 locally in psoriatic keratinocytes or cancer cells that have a vitamin D receptor has the advantage of attaining very high intracellular concentrations of 1α,25(OH)2D3, thereby giving the hormone the opportunity to downregulate the cells' growth for at least several days (Smith et al., 1988Smith E.L. Pincus S.H. Donovan L. Holick M.F. A novel approach for the evaluation and treatment of psoriasis: oral or topical use of 1,25-dihydroxyvitamin D3 can be a safe and effective therapy for psoriasis.J Am Acad Dermatol. 1988; 19: 516-528Abstract Full Text PDF PubMed Scopus (177) Google Scholar;Bower et al., 1991Bower M. Colston K.W. Stein R.C. Hedley A. Gazet J.C. Ford H.T. Combes R.C. Topical calcipotriol treatment in advanced breast cancer.Lancet. 1991; 337: 701-702Abstract PubMed Scopus (113) Google Scholar;Hoech et al., 1994Hoech H.C. Laurberg G. Laurberg P. Hypercalacemia crisis after excessive topical use of a vitamin D derivative.J Intern Med. 1994; 235: 281-282Crossref PubMed Scopus (30) Google Scholar). The localized increase in the concentration of 1α,25(OH)2D3 within the hyperproliferative cells is not likely to have any systemic effects such as hypercalcemia. The proliferative cells may also be less prone to developing a resistance to the therapeutic effectiveness of 1α,25(OH)2D3 if it is produced in sustained higher levels inside the cell. We appreciate the generous gift of the confocal microscope from Mr. Donald Christal and California Sun Care Inc., Los Angeles, CA, and thank David Jackson for his help with the graphics. This work was supported in part by NIH Grant AR36963.