Cultured Human Fibroblasts and Not Cultured Human Keratinocytes Express a PTH/PTHrP Receptor mRNA

Nancy M. Hanafin, * Tai C. Chen ,* Gerhardt H einrich ,t Gino V. Segre,:l: and Michael F. Holick* Vitamin D, Skin, and Bo ne R esea rch Labo rato ry, Sections of *Endocrino logy and '[ Molecular Medicine, Departm ents of Medjcine and Physiology, Boston University Medical Center; and :j:Endocrine Unj t, D epartm ent of Medicine, M assachusetts General H ospital,

H arva rd Medical School, Boston , Massachusetts, U.S.A.

There is increasing evidence that parathyroid hor­mone (PTH) and PTH-related peptides (PTHrP) are involved in normal skin cell growth; therefore, we investigated whether the PTH/PTHrP receptor was expressed in cultured human keratinocytes and der­mal fibroblasts. Northern analyses of poly (A)+ RNA isolated from cultured fibroblasts revealed two PTH/ PTHrP receptor transcripts with one major band at 2.5 kb and one minor band at 2.3 kb. These tran­scripts were consistent with those found in human osteosarcoma cells, which are known to express PTH/ PTHrP-R mRNAs. In contrast, after repeated North­ern analyses no PTH/PTHrP receptor transcripts were found in poly (A)+ RNA isolated from cultured keratinocytes. Reverse-transcriptase/nested poly­merase chain reaction analyses of total RNA isolated

P ara. th.yroid .hormo. ne-r.·elated. peptide's (PTHrP) bioac­tivity was initially identified in tumors associated with humoral hypercalcemia of malignancy [1 ,2]. T he 141 amino acid PT HrP shares 70'% homology with PTH in its first 13 amino acids but diverges completely in its

primary structure thereafter. Studies using synthetic PT HrP N­termina l fi·agm ents have demonstrated tha t fragments that bound to the PTH receptor have caused biologic responses similar to PTH in cultured bone and kidney cells [3). Several groups have postulated that a single receptor species, at least in bone and kidney cells. m ay m ediate many physiologic fun ctions of both PTH and PTHrP [4-6). A human PTH/ PTHrP receptor eDNA encoding a 593 amino acid protein has been cloned [7] . Using a eD NA probe for thls PTH/PTHrP receptor, Urena e/ a/ [8) demonstrated PT H / PTHrP receptor transcripts in many nonclassical PTH target organs and tissues, including the skin. T wo equally abundant PTH/ PTHrP receptor transcripts were present in poly (A) + R.N A isolated fro m whole rat skin ; a 2.3- 2 .5-kb transcript, identical to that fo und in bone and kidney and a small er 2.0 - 2.2-kb transcript. T he data support early e li.']Jet;mentation that suggested PT H receptors w ere present in skin fibroblasts [9).

Manuscript received December 17, 1994; final rev ision received February 17, 1995; accepted for publicatio n March 3, 1995.

R eprint requests to: M . F. 1-lo li ck, Vitamin D , Skin, and Bo ne R esearch Laboratory, Boston University Medical Center, 80 East Concord Street, M-1 013, Boston , MA 02118.

from cultured keratinocytes and fibroblasts con­firmed the Northern analyses data that the PTH/ PTHrP receptor was expressed in cultured fibroblasts but not in cultured keratinocytes. When cultured fibroblasts and keratinocytes were exposed to 10- 7 M PTH (1-34) there was a twofold increase in cAMP levels in the fibroblasts and no demonstrable increase was noted in keratinocytcs. These results suggest that skin fibroblasts possess the classical PTH/PTHrP re­ceptor and are target cells for PTH and PTHrP whereas keratinocytes do not have the receptor and are unresponsive to its N-terminal agonist in the stimulation of cAMP formation. Ke]l 1vords: cAMPI nested PCR/3T3 fiiJroblasts. ] ln11est D emwtol 105:133-137, 1995

A variety of nonnal tissues syn thesize and secrete PTHrP [3] . In fact, th e presence of PTHrP bioactivity in nonmalignan t cells was first dem onstrated in conditio ned medium harvested from conflu­ent human keratinocytc cultures [1 0). However, the physiologic ro le and the functional pathway of this peptide in n orm al skin is still unclear , although some have suggested that PTHrP m ay fi.tn ction as an autocrin e or paracrine factor [3, 11 J. In 11itro and i11 11i11o studies in our labo ratory have demonstra ted that PTH (1-34) and PTHrP (1 -34) are po ten t inhibitors of epidermal cell proliferation and PT.H (7-34) , an antagonjst of the PTH/ PTHrP receptor, can block the antiproliferative effect of PTH (1 -34) and stimulate epiderma l proliferation in cul tured keratinocytes [12,13). Additionally, Kaiser et a/ [14) , after expressin g an antisense PTHrP sequence in an established human kera tinocyte cell line, demonstrated enhanced keratinocyte ce ll gmwth.

ln considering these respon ses of keratinocytes to PTH and PTHrP, we wondered if the PTH/ PTHrP receptor. p resum ably at least one m ediato r for th e bio logic expressio n of both the PTH and PT.HrP hormones, w as presen t in cultured human keratinocytes and fibroblasts. Because PTH stimul ates the fom1ation of cyclic aden­osine monophosphate (cAMP) in ta rget tissues l,l110wn to express the receptor, we m easured intra cellular cAM P levels in keratino­cytes and fibroblasts after treatm ent with PTH (1-34). To deter­mine mR.NA expression , w e also performed N or them ana lyses on poly (A) + from cultured keratinocytes and fibroblasts usin g a eDNA that codes for a common human PTH/ PTHrP receptor [15) . Additionally, w e perform ed reverse transcriptase (RT) and nes ted

0022-202X/95/S09.50 • SSDl0022-202X(95)00160-M • Copyright © 1995 by T he Society for Investigative Dermatology. Inc.

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134 HANAFIN f::T AL

polymerase chain reaction (PCR) analyses on total RNA isolated from human keratinocytes and both human and mouse fibroblasts.

EXPERIMENTAL PROCEDURES

Cell Culture Keratinocytes were grown in culture using a modification of Rheinwald and Green [1.6]. Keratinocytes were obtained from neonatal foreskins and plated on a lethally irradiated 3T3 feeder layer in a serum-free MCDB 153 basal medium con­taining 0.15 mM calcium and supplemented with growth factors, including bovine pituitary extract, 3 ~J.g/ml; epidermal growth factor, 25 ng/ml; insulin, 5 ~J.g/ml; hydrocortisone, 200 ng/ml; cholera toxin , 0.1 !J.glml, and prostaglandin E 1, 50 ng/m] [17]. Cells were fed and maintained in the same medium without cholera toxin and hydrocortisone three times per week. Second-passage keratinocytes grown on 1 00-mm or 35-mm dishes were used for Northern ana.lyses or cAMP determinations, respectively. To avoid the potentia l contamination of 3T3, some primary cultured kerati­nocytes were grown without a 3T3 feeder layer for reverse­transcriptase/polymerase chain reaction (R T /PCR) analyses. Nor­mal fibroblasts were grown from buman foreskins in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum as previously described [1 8]. For RT /PCR experiments, cells were harvested from single 1 00-mm dishes. Total RNA from human osteoblast- like osteosarcoma cells (SaOS-2) for both North­em and R T /PCR analyses was extracted and purified as described [15].

cAMP Determination For cAMP experiments, keratinocytes and fibroblasts were plated on 35-mm dishes. Growth factors were removed from the keratinocyte media and fetal bovine serum was removed from the fibroblast media 2 d before the e"-"(Jeriment. At the experiment's initiation, cells were preincubated for 30 min at 37°C in 0.5 ml of fresh MCDB-153 or DMEM medium (Sigma, St. Louis, MO) for keratinocytes and fibroblasts , respectively, foLlowed by treating the cells with human parathyroid hormone (hPTH 1-34) (Bachem, Torrance, CA) at a final concentration of 10- 7 M or vehicle (phosphate-buffered· saline) only. Following incubation for 10 min at 3 rc, the reaction was terminated by removing the medium and adding 0.5 ml of 0.1 N NaOH. The resulting cell suspension was neutralized with 1 N HCI and aliquots were quantitated using a commercial cAMP assay kit (Amersham, Ar­Lington Heights, IL). Standard error of the mean was calcu la ted from triplicate determinations.

Northern Analysis Total cellular RNA was prepared fi:om keratinocytes and fibroblasts using a modification of the guani­dinium thiocyanate-cesium chloride technique [19]. Total RNA from mouse kidney was prepared in a similar marmer. Poly (A) + RNA was further isolated from keratinocyte and fibroblast by utilizing a PolyATtract mRNA Isolation System (Promega, Madi­son , W I). Tota l RNA and poly (A) + samples were denatured and fractionated on a 1%/3 7% agarose/formaldehyde gel containing ethidium bromide. An RNA ladder (Gibco/BRL, Grand Island, NY) was also run to determine message size at the hybridization bands. The RNA was transferred via capil lary action to a nylon membrane, Hybond N (Amersham) and cross-linked to the mem­brane 11ia an ultraviolet transilluminator (lBL) . Membranes were prehybridized at 42°C for 2 h in a solution containing 50% formamide, 100 !J.g/ml denatured nonhomologous DNA, 5 X SSC, 0.5% sodium dodecyl sulfate, 10 X Denhardt's, fifty millimoles per liter of Na2P04 , and 10% dextran sulfate (Sigma). A eDNA (Genbank accession number, L04308) encoding a humru1 PTH/ PTHrP receptor [15] was labeled with [a32P] dCTP (Amersham) via the Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA) . A Sephadex G-50 column (Boehringer Mannheim Corp, Indianapolis, IN) was used to remove unincorporated free nucleotide. All blots were stripped and rehybridized with a human glycera ldehyde-3-phospbate dehydrogenase (GAPDH) oligonucle­otide (Oncogene Science, Uniondale, NY), whose sequence was based on the eDNA . sequence cloned by Tso and colleagues [20].

TI-lE JOURNAL OF INVESTIGATIVE DERMATOLOGY

The oligonucleotide was 5 ' labeled with [y 2 P] ATP (Amersham). A Sephadex G-25 column (Boehringer Mannheim Corp) was used to remove unincorporated free nucleotide. Hybridizations took place overnight at 65 °C in a solution containing 1 M NaCI, 50 mM Tris-HCI (pH 7.5), 10% dextran sulfate, 1% sodium dodecyl sulfate, and 100 !J.g/ml denatured nonhomologous DNA (Sigma). Autoradiography of blots was carried out at -70°C using Hyper­film (Amersham) and an intensifying screen (Dupont/NEN, Bos­ton, MA) .

RT/Nested PCR Analysis Total RNA aliqu ots (1 p,g) isolated from cu ltured lmman keratinocytes, grown either with or without a 3T3 feeder layer, human fibroblasts, human osteoblast-like osteosarcoma cells (SaOS-2), and immortalized mouse fibroblasts (3T3s) were added to separate reverse transcriptase (RT) reactions [21]. The buffer consisted of7.0 mM MgCI 2 , 50 mM KCI, 10 mM Tris-HCI (pH 8.3), deoxynucleotide triphosphates at 1 mM each, 1 unit RNase inhibitor, 2.5 !J.M random hexamers, and 2.5 units of cloned Moloney Murine Leukemia Virus (M-ML V) Reverse Tran­scriptase (Perkin-Elmer Cetus/ Applied Biosystems, Foster City, CA). The final volume for each reaction was 20 !J.l. The reaction mixtures were incubated at room temperature for 10 min, 42°C for 45 min, 99°C for 5 min, and soc for 5 min. PCR amplifications were carried out in the Gene Amp PCR system 9600 (Perkin-Elmer Cetus). Amplifi cations were done in a final buffer volume of 100 !J.I consisting of 2 mM MgCI 2 , 50 mM KCI, 10 mM Tris-HCL (pH 8 .3), and 2.5 units AmpliTaq DNA Polymerase (Perkin-Elmer Cetus). PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using 1 0 !J.M of the forward primer 5'-TCCCATCACCATCTTCCA-3 ' and 10 !J.M of the reverse primer 5 ' -GTCCACCACCCTGTTGCT -3'. PCR conditions were 95°C for 2 min, followed by 35 cycles of95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds with a final extension at 72°C for 5 min. Nested PCR [22] was used for the PTH/PTHrP-R. The outer forward primer 5'-GGGCACCAGGTGAAGTGGT-3 ' and the outer reverse primer 5 '-GGTTGCTCTGACAC­CGACCC-3' were used for the first amplification. PCR conditions were 95°C for 2 min, followed by 30 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds with a 3-second extension per cycle. Final extension was at 72°C for 5 min . For the second amplification , 5 !J.I of the first-round amplification mixture was added to the same mixtu.re as in the previous amplification but this time the inner primer concentrations were doubled to 20 !J.M rather than 10 !J.M. T he inner forward primer was 5'-CGGGAG­GTAmGACCGCCTAG-3' and the inner reverse primer was 5 '-CAGAA TCCAGT AGTAGTTGG-3'. PCR conditions were the same as for the first amplification except cycle duration was 25. Samples of 20 !J.I of PCR reaction products were analyzed by electrophoresis on a 2% agarose gel. Bands were visualized by eth.idium bromide staining. At some amplifications [ a 32P] dCTP (Amersham) was added to reaction mixtures and gels were dried and exposed to Hyperfilm (Amersham). The PCR amplification products for PTH/PTHrP-R were further analyzed for authenticity by restriction enzyme digestion with Fok I, Bsg I, and Drd I (New England Biolabs, Beverly, MA).

RESULTS

Stimulation of cAMP Production in Fibroblasts Treated with hPTH (1-34) The ability of a PTH N-terminal fi·agment to stimulate cAMP production was studied in cultured nom1al human keratinocytes and dermal fibroblasts. Treatment of fibroblasts with 10 - 7 M of hPTH (1-34) caused a twofold increase in cAMP levels over the basal level (4.2 ::!:: 0.1 pmolldish versus 2.0 ::!:: 0.05 pmolldish). The data were significantly different and p was < 0.005. In contrast, when keratinocytes were treated with 10- 7 M ofhPTH (1-34) no stimulation of cAMP formation was observed. Treated keratinocytes were 4.0 :::':: 0.2 pmol/dish and untreated keratino­cytes were 3. 9 ::!:: 0.1 pmol/ dish. T here was no significant difference between these data points. Because hPTH (1-34) stimulated cAMP

VOL. 105, NO. l JULY 1995

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PTH/PTHrP-R 2.5 Kb •

1 2 3 4 5

GAPDH. B 1 2 3 4 5

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GAPDH • PTH/PTHrP-R •

1 2 3 4 5 6 7

formation in cultured fibroblasts but not in cultured keratinocytes , we exa mined whether cultured human fibroblasts and keratino­cytes possessed the same PTH/ PTHrP receptors as those found in kidney and bone cells.

Northern Blot Analysis Utilizing the recently cloned, full­length human PTH/PTHrP receptor eD NA [15], Northem ana lysis was performed on poly (A) + RNA from cultured human keratino­cytes and fibroblasts. In all hybridization experiments, approxi­mately 10 }.Lg total RNA from mouse kidney cells and / or human osteoblast-like osteosarcoma cells (SaOS-2) were used as positive controls. Lane l of Fig lA contained the positive control SaOS-2 cells. Lane 2 contained 5 J.Lg of poly (A) + RNA fi:om keratinocytes. No signal was visible in this sample . Lanes 3 and 4 contained SaOS-2 and mouse kidney total RNA, respectively. As can be seen, a highly expressed 2.5 kb PTH/ PTHrP receptor mRNA was found in both SaOS-2 and mouse kidney cells . Also visib le in both samples was a less prominent transcl;pt at 2.3 kb. Interestingly, three smaller transcripts were expressed i11 the SaOS-2 bone cells . La11e 5 contained 5 J.Lg of poly (A) + RNA isolated from fibrobla sts . The major 2. 5-kb transcript was cleady expressed as well as another at 2.3 kb . No smaller transcripts w ere observed even after pro­longed exposure of the autoradiograph . Ethidium bromide staining of the gel and rehybridization of the membrane with an oligonu­cleotide specific for human GAJ>DH confirmed sampl e integrity and relative loading concentrations. In Fig 1A, the GAPDH signals were visible in all of the lanes except for lane 4, tl1e mouse kidney cells. Because the GAPDH oligonucleotide probe ·was specific for hum an cells, no detectable signal was expected. Apparent and exp~cted w ere the int~nsities of the GAPDH sig11als in the poly (A) RNA fi·om keratmocytes and fibroblasts as compared to the GAPDH sig11als from SaOS-2 total RNA. In a comparison hybrid­ization experiment, five times the amount of poly (A) + RNA from keratinocytes as from fibroblasts still did not show PTH/PTHrP-R expression , even with prolonged exposure of the autoradiograph (data not shown). In a total of five separate analyses the PTH/ PTHrP receptor m essages were consistently found in poly (A) + RNA samples prepared from cultured dermal fibroblas ts but not from cultured keratinocytes .

RTINESTED PCR Analysis R everse transcriptase and nested PCR was used to analyze total RNA isolated from human kerati­nocytes and fibroblasts as well as from mouse immortalized fibro­blasts (3T3s) and from human osteosarcoma cells (SaOS-2). In these experiments, the success of th e eDNA synthesis from the RT

PTH/PTHrP RECEPTOR mRNA IN FIDR O BLASTS 135

Figure 1. PTH/PTHrP-R expression in cultured human fibro­blasts as shown by Northern analysis and RT/nested PCR analysis. A) Northern blot analysis of total RNA prepared from SaOS-2 (la ues 1 ami J) and mouse kidney cells (lau e 4) and poly (A) + RNA from kcratinocytes (lau e 2) and fibroblasts (laue 5). The blots were hybridized with the full-l ength eDNA encoding the human PTH/ PTHrP receptor. B) Laue 1 was RT and GAPDH PCR amplification of to tal RNA from human keratinocytes cultured without a 3T3 feeder layer. L aue 2 was an aliquot of these same cells after RT and nested PCR amplification with PTH/ PTHrP-R primers. Laues 3 through 6 arc RT/ nested PCR samples from human keratinocytes cttltured with 3T3 cells, human fibroblasts. human osteoSilrcoma cell s, a11d 3T3 cell s, respectively. Lane 7 is the nested PCR product from the human PTH / PTHrP receptor eDNA .

reaction was verified by amplifying an aliquot of the R T product w ith pdmers for the GAPDH constitutive gen e and confirming by gel electrophoresis the presence of the predicted PC R product. For PTH/ PTHrP analyses, parallel first-amplification reactions were performed on eDNA aliquots and PCR products were analyzed by gel electrophoresis. T he first amplification of a human PT H / PTHrP receptor eDNA produced the predicted 675-bp product and the second amplification produced the predicted 360-bp product. Nested PCR analyses of RT products from human keratinocytes that w ere initially cultured on a 3T3 feeder layer, as well as from human fibroblasts , mou se 3T3 fibrobl asts , and human osteosar­coma celJs demonstrated identical results to th ose from cl1c human PTH/ PTHrP receptor eDN A . In contrast, nested PC R ana.l yses of RT products from human keratinocytes that were cultured entirely without a 3T3 feeder layer (Fig lB) demonstra ted neither a first nor second PCR amplifi cation product. Lan e 1 con tained the R T/GAPDH-amplified PCR. product from keratinocytes grown ~i~hout a 3T3 feeder layer. T he p1·edicted 762-bp product is clearly VISible. Lm1e 2 contained an identical aliquot from this R T reaction tha.t was amplified via the n es ted PCR protocol previously de­scnbed. No PCR products were visible. Lane 3 contain ed an RT / nested PCR sm11ple from keratinocytes that were cultured w ith a 3T3 feeder layer. Lanes 4 tlu·ough 6 were R.T/nested PCR samples fl·om human fibroblasts, human osteosarcoma cells , m1d mouse 3T3 fibroblasts, respectively. Lane 7 contain ed th e nested PCR product from the humm1 PTH/ PTHrP receptor eDNA. As can be seen, lanes 3 through 7 displayed identical second-amplifi­cation PCR products of the predicted 360 bp size. Firs t-am plifica­tion PCR products of 675 bp were also identical for these samples and parallel amplifications with GAPDH primers produced th e predicted product (data not shown). R estriction enzyme digests of the amplification products with Fok I, Bsg I, and Drd I further confirmed their authenticiry.

DISCUSSION

Aliliough we [12,13] have demonstrated that specific PTH and PT HrP N-terminal fragments arc antiproliferative agents in cu l­tured human keratinocytes and in mouse epidermis i11 v ir10, North­ern analyses indicated the PTH/ PTHrP receptor mRNA was not expressed in cu ltured keratinocytcs. Jn contrast, cultured dermal fibroblasts expressed two PTI-I / PTHrP receptor transcdpts with one major band at 2.5 kb and one minor band at 2.3 kb. T he transcripts observed in fibroblasts w ere consistent with those seen m hybridizations of RNA isolated fi·om SaOS-2 cells and mouse

136 HANAFIN ET AL

kidney cell s, w hich are known to be PTH/PTHrP receptor positive [8] . The presence of two transcripts m ay be due to alternative transcrip tion start sites, spl icing, or the presence of closely related ml"t.NAs. To in crease detectio n sensitivity for the PTH/PTHrP transct"ipt, we uti lized RT/nested PCR. T he resu lts confi rmed the Northern ana lyses data and clearly showed that the PTH/PTHrP receptor m essage was present in hum an fibro blasts, human osteo­sarcoma cells and mouse 3T3 cells. In terestingly, the enhanced sensitivity of R.T / nested PCR revealed the PTH/PTHrP recepto r in kera tinocytes cul tured on a 3T3 feeder layer. Because we found the PTH/PTHrP receptor in 3T3 cells, it was thought that RT I PCR amp Lification of RNA isolated fi:om keratinocytes grown on a 3T3 feeder layer co uld generate receptor-positive amplifi ca tion products . RT/nested PCR analyses of keratinocytes that were cultured witho ut a 3T3 feeder layer demonstrated no PTH/PTJ-IrP receptor, suggesting th at the product observed was derived fi·om the 3T3 cells of the feeder layer and not from the keratinocytes. The data are consistent w ith the additional observatio n that in tra­cellul ar cAMP levels increased when fibrobla sts were treated with PTJ-1 (l-3 4) (10 - 7 M), but in identically treated kera tinocytes there was no detectable cAMP response.

Because the re is no detectab le PTJ-1/PTJ-IrP receptor in human cul tured ke ratinocytes, the data raise the possibility that PTHs and PTHrPs antipro liferative effects on kera tinocytes ma y be mediated by PTJ-1/PTHrP receptors located in adjacent fibroblasts. Such a pathway is believed to be one way PTH increases osteoclastic bone resorpt ion. Because mature osteoclasts do not have PTH receptors, PTH m ay first act on osteoblasts and/ or stromal fibroblasts to in crease bone resorption [23]. M oreover, paracrine- and/or auto­ct"ine-mediated effects on skin cell s by PTHrP are also possible .

It is also possible that there are differen t receptors for PTH/ PTHrP in keratinocytes and fibroblasts. T his idea is strengthened fro m the va riability in the biologic potency of difterent PTH/ PTHrP analogs in numerous tissues [3] . Studies of the biologic effects of several PTJ-I rP fragm ents in different cell types have suggested that the re m ay be a PTHrP receptor distinct from the classic PTH/PTHrP receptor, which is coupled to adenylate cyclase through th e G-protein (7] . For example, recombinant PTHrP (1.-141) and PTHrP (1-108) were two to four times more potent than PTHrP (1-34) in the stimulatio n of plasminogen activator activity in UMR cell s [24]. Simihrly, hPTHrP (1-34) and bPTHrP (1-108) were not active in inhibiting bone resorption, w hereas th e carboxy-terminal fragment hPTHrP (107-139) was a potent inhib­itor of bone resorption (25]. Such data suggest that a distinct PTHrP receptor(s) m ay recogni ze specific fragments.

Similarly, other studi es suggest that particular PTHrP fragments may induce different signal-transduction mechanisms. Specifica lly, O rloff et a/ [26] using sq uam ous carcinoma cells demonstrated that hPTHrP (67-86)NH2 or hPTHrP (1-141) ca used a rapid and trans ient rise in intracel!uJar ca lcium in a dose-dependent manner between 10- 11 M and 1 o-s M but there was no stimulation of cAMP formation detectable even with 10 - 6 M hPTHrP (67- 86). In a separate study, O rloff eta/ [27] suggest hu man ca rcinoma cells and normal keratinocytes express mR.NAs re lated to but distin ct from the human PTH receptor mRNA. If there are distinct receptors and presumably specific PTH and PTHrP fragment activation sites o n these receptors, then this could explain no cAMP formation by PTH (1-34) and no PTH/PTHrP receptor expression in keratino­cytes.

T hroug h cAMP determinations, North ern anal yses, and RT I nested PCR analyses, this study localizes the human PTH/PTHrP receptor in fibrob lasts but not in keratinocytes. Numero us ques­tions remain , however, as to wheth er the bio logic activities ofPTH and PTI-IrP in skin cells are m ediated through paracrine and/or autocrine pathways o r w hether identical m· distinct receptors exist. More resea rch is necessary to understand the biologic activation o f endogeno usly occurring PT H and PTHrP in skin cells and the potentia l the rape utic use for these hormo nes in regulatin g skin cell growth and dilferenti:1tion.

THE J OU RNAL OF INVESTIGATIVE DERMATOLOGY

T his "'ork wns s11pported i11 pnrt by N IH grnllts DK43690 nllll MO IRR00533. We thn11l' David M. jnckso11 }or his excel I ell/ .grnpltics nss isln11cc 11•itlt rite mn111t.<cript.

REFERE N CES

I . Sccwart AF, Horst R, Deftos LJ , Cadman EC, Lang R . Broadus AE: Biochemical cv:1luation o f paLicnts with c:tn ccr-a ssociatcd hypercalcemia: e vidence for humoral and nonhumoral gro ups. N Eux / J Ivied 303: 1377-1383, '1980

2. Strcwlcr GJ, W illi ams R.D , NisscnsonltA: 1-hnuiln rcnn l c:u cinorna cel ls produce hypercalcem ia in the nude mo use and a novel protei n recognized by parathy­roid hormone receptors. J Cliu ft iiiW 7 1:769-774, 1983

3. Orloff JJ, Wu TL. Stewart AF: Parathyroid ho rmone- like protein s: biochemical respo nses and receptor inLeractions. f:.'mlm:r Rei' I 0:476-495, 1989

4. Juppncr H . Abou-Samra All, Uneno S, Giu WX, Potts JT J r , Segre GV: The parathyroid hormo ne like peptide associaLcd with humoral hypercalccntia of nwlignancy and par:1thyroid ho rrno ne bind to the same receptor on the plasma membran e of ROS 1712.8 cells. J Bioi Che111 263 :8557-8560, 1988

5. N issensonllA, KarpfD, Bambino T, Winer J . C anga M. Nyiredy K, Arnaud CD: Cova lent labeling of a high-atli nity guanyl nuclcotidc-scnsiLivc para thyro id hormo ne receptor in canine renal cortex. Biochemistry 26:1M74-1878, 1987

6. Fukayama S, Bosma TJ , Goad DL, Voelkel EF, Tashjian AH J r: Human parnthyroid lton-rto ne (PTH)-rclated pro tci11 and human PTI-1: comp~1r:.ttivc bio logical acrivities on human ho ne cc iJ s and bo ne resorption . Eudocritwlog}1

123:2841-2848, "1988 7. Juppner H. Abou-S:unra AB . Free man M. Ko ng XF, Schipani E. Richards J.

Kolakowski LF, Hock J . Potts JT Jr, Kronenberg HM. Segrc GV: A G protein-lin ked receptor for parathyro id hormone :md parathyroid hormone­related peptide. Scit·ucc 254: I 024 - 1026. 199 1

8. Urena P, Kong XF, Abou-Samra All, Juppner 1-1, Kronenberg HM. Potts JT Jr. Scgre GV: Parathyroid honnonc (IYT I-1)/PTH-rclated peptide receptor mes­sage dbonudcic :1cids arc widely dislributed in rat tissues. Emiocritwlogy 133:617-623, 1993

9. Goldrin g Slt, Mahaffey J E. R.osenblatt M, Dayer JM . Potts JT Jr. Krane SM: Parathyroid honn o nc inhibitors: compo1rison of bio logic:JI activ ity in bon e and skin derived tissue.) C li11 E11docriuo/ Mern lt 48:655-659. 1979

'10. Mercnd.ino JJ Jr. Insogna KL. Milstonc LM , Broadus AE. Stewart AF: Cultured human kcratinocytcs prod uce a parathyroid hormone-li ke prorcin . Science 23 1:388 - 390, 1986

11. OrloWJJ. W u TL, Stewa rt AF: Parathyroid hormone-like protein s: biochemical responses and receptor in ter;Jcdons. Em/ocr l~t?v 10:476-495, 1989

12. Ho lick MF, Nussbaum S. Persons KS: PTH-Iike humoral hyperca lcemia f.1ctor (1-IH F) of malignancy m:.ty be an epidermal diffCrcntiarion f.1ctor: synthetic hHI-IF (1-34)N I-12 inhibits proliferatio n and induces tcnninal diJlCrentiation of cul tured human kcratinocytcs. J Rom: J\ll iuernl Res 3:S2 14, 1988

13. Ho lick MF, R ay S. C hen TC, Tian X , Persons K: A parathyroid hormone antagon ist stimul ates cpidennal prolifCr:.1tion and lwir growth in mice . Pror Nt~tl Jlrnd Sci USA 91:8014-80'16, 1994

14. Kaiser SM, Lan cuvillc P. Bernier SM. R . .hirn JS, Kremer R., Golrzman D : Enhanced g rowth of a hun1an keratinocytc ce ll line indu ced by nntiscnse RNA fo r parathyroid honn one-rela ccd peptide.) Uiol C ltct/1 2(,7: 13623-'13628. 1992

IS. Schi pani E, Karga H. Karaplis AC, Potts JT J r. Kronenberg HM . Scgrc GV. Abou-Sa rnra AD . Juppncr 1-1 : Identi cal complementary dco:\.-yribon uclc ic acids encode a huma n renal nnd bo ne para th yroid hormone (PTJ-1) /PTI-I-re lated peptide receptor. E~tdocrilwlo.~ )' 132:2 157-2 165 . 1993

16. Rhcin wa ld JG. Green 1-1 : Form ation of a keratiniz in g epithe lium in culture by ::1

cloned cellliue deri ved fro m a teratoma. Cell 6:331-344. 1975 17. Smith EL, Wal worth NC, H o lick MF: Effect of1", 25-dihydroxyvitamin D, on

rit e morplt o logic and biochemical differentiation of cultured human epidermal keratinocytcs g rown in serum-free conditio ns. J l11rlcst 01:nuatol 86:709 -714. 1986

18. C lemens TL. AdamsJS. Horiu chi N . Gilchrest BA. C ho 1-1. Tsuch iya Y. Matsuo N. Sudn T. HoUck MF: ln tcracLion of 1.25-dihydroKyvitamin D3 ·with kcr;1tinocytcs ;utd fibrob lasts from skin of normal subjects and a subject with vitamin D-dependcnt ri ckets, type II : ;1 model for Lhe study of the mode of actiou of 1.25-dih ydroxyvirarnin D3 .) C/iu Eudoni11ol Mcrn/1 56 :824-830, 1983

19. ChirgwinJ , Przybyla A, MacDonald R .. Ru tter W: Isola tion of biologica lly active ribo nucleic acid from sources enriched iu ribonuclease . Bioc!Jemistl1' 18:5294 -5299, 1979

20. TsoJY, Suu X-1-1, Kao T-1-1. Reece KS, W u R: Isolatiou and characterizaLion of rat and human g lyccra ldchyde-3-phospha tc dehydrogenase cON As: geno mic complexity and mo lecular evolutio n of the gene. N ucleic Acids Res 13:2485-2502, 1985

2 1. Wang AM, Doyle MV. Mark OF: Quantitation of mR.NA by the po lymerase chai n reaction . l'roc Na t/ I Icari Sci USA 86:97 17-9721. 1989

22. Garson J A. Tedder RS. Briggs M. Tuke P, Glazebrook JA. Trute A, Parker D, Barbara JAJ, Contreras M. Aloysius S: Detectio n of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lnuccr 335:14 19-1422, 1990

23. Holick MF. Krane SM. Potts JT Jr: Calcium. phosphorus and hone metabolism. In: W ilson JD (ed .). HMrismt 's Pri11ciplcs of lu tcma! J\IIcdiriuc1 ·t2til cd. 1888- '1 889. McG raw Hill , Inc, New York, 199 1

24. Hammonds R G Jr. McKay P, Winslow GA, Diefenbach-Jagger H . Gri ll V, Glatz J, Rodda C P, Wood W I, Martin TJ: Purification and char:1ctcrization of recombinan t human parathyroid ho rmone- related protein. J BitJI Chem 264: 14806-148 1'1. '1989

VOL. 105. NO. JULY 1995

25. Fen ton AJ. Ke mp OE, Kent G N . Moseley JM. Z hcng MH. Rowe OJ. Brirto J M. Martin TJ , N icholson GC: A carboxyl-[Crminal peptide from the p;lr:t rltyroid

IJOnllOne-rcl:ttcd pro tein inhibits hone resorp tion by ostcoclasts. E,uJocriuo/JJ.':: )I 129: 1762- 1768 , 199 1

26. O rlo ff JJ , Ka ts Y, Milnick M , Gasall a-H erraiz J. !sales C M : Evidence for a recepto r o n squ am o us c~1 rc inoma cell lines w hich recogni zes a mid-region

PTH / PT H rP lliCEPTOR. m R.NA IN FIUROBLASTS 137

ti-agnh:n r of p:lrath yroid ha n none-related protein , PTHrP (67-86)NH 2.J Bone

Millc•m/ Rl's 8:5 133, 1993 27. O rl o ff JJ , Urena 1'. Schipani E. ll ibaudo AE. Milstone LM. Philbrick WM.

Abou-S:~m ra A l3 , Scgrc GV, Juppm!l- l-1 : Human squ:u no us carci no ma cells a.nd

normal kcrati nocytcs express ml"t.NA, related lO but distin ct fro m huma.n PTH

receptor m R NA. J /Jo11c Jllilll'l'lli Res 7:5230, 1992