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PTHrP Regulates Epidermal Differentiation in Adult Mice

1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1998.00428.x

1523-1747

John Foley, John J. Wysolmerski, Barbara E. Dreyer, Arthur E. Broadus, William M. Philbrick, B. Jack Longely,

Fibroblast Growth Factor Research

Emerging evidence suggests that parathyroid hormone-related peptide (PTHrP) serves as a regulator of the development and/or differentiation of a number of organs, including endochondral bone, the tooth, and the mammary gland. Although disruption of the PTHrP gene by homologous recombination results in a lethal chondrodystrophy, PTHrP-knockout mice that have been rescued by the transgenic replacement of the peptide in cartilage display abnormalities in ectodermally derived structures including the skin. At 6–8 wk of age, these rescued PTHrP-knockout mice displayed a markedly thinned epidermis and striking hyperkeratosis, hypoplastic sebaceous glands, and a fibrotic dermis. In contrast, transgenic mice that overexpress PTHrP by virtue of the human keratin-14 promoter displayed a thickened ventral epidermis with marked acanthosis and papillomatosis, hyperplastic sebaceous glands, and a cellular dermis. The absence of PTHrP appeared to result in the reduction of the basal keratinocyte compartment and premature acquisition of suprabasal and granular differentiation markers, whereas overexpression of the peptide generated reciprocal findings. No difference in the epidermal proliferation rate was found in PTHrP-null skin and although an increase was observed in keratin 14-PTHrP transgenic animals, their epidermis did not express the hyperplasia marker K6. Finally, the replacement of PTHrP in the basal keratinocytes of rescued PTHrP-knockout mice under the direction of the keratin 14 promoter reversed the abnormalities seen in PTHrP-null skin. These findings suggest that PTHrP regulates the rate of keratinocyte differentiation in the skin of adult mice. Emerging evidence suggests that parathyroid hormone-related peptide (PTHrP) serves as a regulator of the development and/or differentiation of a number of organs, including endochondral bone, the tooth, and the mammary gland. Although disruption of the PTHrP gene by homologous recombination results in a lethal chondrodystrophy, PTHrP-knockout mice that have been rescued by the transgenic replacement of the peptide in cartilage display abnormalities in ectodermally derived structures including the skin. At 6–8 wk of age, these rescued PTHrP-knockout mice displayed a markedly thinned epidermis and striking hyperkeratosis, hypoplastic sebaceous glands, and a fibrotic dermis. In contrast, transgenic mice that overexpress PTHrP by virtue of the human keratin-14 promoter displayed a thickened ventral epidermis with marked acanthosis and papillomatosis, hyperplastic sebaceous glands, and a cellular dermis. The absence of PTHrP appeared to result in the reduction of the basal keratinocyte compartment and premature acquisition of suprabasal and granular differentiation markers, whereas overexpression of the peptide generated reciprocal findings. No difference in the epidermal proliferation rate was found in PTHrP-null skin and although an increase was observed in keratin 14-PTHrP transgenic animals, their epidermis did not express the hyperplasia marker K6. Finally, the replacement of PTHrP in the basal keratinocytes of rescued PTHrP-knockout mice under the direction of the keratin 14 promoter reversed the abnormalities seen in PTHrP-null skin. These findings suggest that PTHrP regulates the rate of keratinocyte differentiation in the skin of adult mice. procollagen II; parathyroid hormone-related protein; receptor, parathyroid hormone/parathyroid hormone-related protein receptor Parathyroid hormone-related peptide (PTHrP) was initially discovered by virtue of its association with the clinical syndrome of humoral hypercalcemia of malignancy, in which PTHrP released from tumors activates classical parathyroid hormone (PTH)/PTHrP receptors in bone and kidney (Broaduset al. 1988). It is now apparent, however, that this is the only circumstance in which PTHrP circulates in quantities sufficient to exert systemic effects and that the primary role of this peptide is as a local paracrine or autocrine factor (Segre, 1994Segre G.V. Receptors for parathyroid hormone and parathyroid hormone-related protein.in: Levine M.A. The Parathyroids. Raven Press, New York1994: 213-229Google Scholar;Broadus and Stewart, 1996Broadus A.E. Stewart A.F. Parathyroid hormone-related protein structure, processing and physiological actions.in: Marcus R. The Parathyroids. Raven Press, New York1996: 259-294Google Scholar;Philbrick et al., 1996Philbrick W.M. Wysolmerski J.J. Galbraith S. et al.Defining the roles of Parathyroid hormone-related protein in normal physiology.Physiological Rev. 1996; 76: 127-173PubMed Google Scholar). Both PTHrP and the PTH/PTHrP receptor have been found to be expressed in a wide array of fetal and adult tissues, andin situ hybridization studies have shown PTHrP mRNA to be expressed typically in a focal pattern in surface epithelia, whereas the receptor mRNA is distributed diffusely in the adjacent mesenchyme (Senior et al., 1991Senior P.V. Heath D.A. Beck F. Expression of parathyroid hormone-related protein mRNA in the rat before birth: demonstration by hybridization histochemistry.J Molec Endocrinol. 1991; 6: 281-290Crossref PubMed Scopus (87) Google Scholar;Urena et al., 1993Urena P. Kong X. Abou-Samra A. Jüppner H. Kronenberg H.M. Potts J.T. Segre G.V. Parathyroid hormone (PTH/PTH-related peptide) messenger ribonucleic acids are widely distributed in rat tissues.Endocrinology. 1993; 133: 617-623Crossref PubMed Scopus (197) Google Scholar;Lee et al., 1995Lee K. Deeds J.D. Segre G.V. Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats.Endocrinology. 1995; 136: 453-463Crossref PubMed Google Scholar). Human keratinocytes in culture were the first normal cells found to secrete PTHrP (Merendino et al., 1986Merendino J.J. Insogna K.L. Milstone L.M. Broadus A.E. Stewart A.F. Cultured human keratinocytes produce a parathyroid hormone-like protein.Science (Wash DC). 1986; 231: 388-390Crossref PubMed Scopus (177) Google Scholar), and immunohistochemical studies have localized the peptide from the basal layer to the granular layer of the epidermis and to the outer root sheath cells in hair follicles (Atillasoy et al., 1991Atillasoy E.J. Burtis W.J. Milstone L.M. Immunochemical localization of parathyroid hormone-related protein in normal human skin.J Invest Dermatol. 1991; 96: 277-280Abstract Full Text PDF PubMed Google Scholar;Wysolmerski et al., 1994Wysolmerski J.J. Broadus A.E. Zhou J. Fuchs E. Milstone L.M. Philbrick W.M. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.Proc Natl Acad Sci USA. 1994; 91: 1133-1137Crossref PubMed Scopus (168) Google Scholar). PTHrP gene expression has been shown to be regulated by a number of agents that influence keratinocyte differentiation, including calcium, vitamin D, serum, and uncharacterized factors produced by dermal fibroblasts in culture (Kremer et al., 1991Kremer R. Karaplis A.C. Henderson J. Gulliver W. Banville D. Hendy G.N. Goltzman D. Regulation of parathyroid hormone-like peptide in cultured normal human keratinocytes.J Clin Invest. 1991; 87: 884-893Crossref PubMed Scopus (137) Google Scholar,Kremer et al., 1996Kremer R. Sebag M. Champigny C. Meerovitch K. Hendy G.N. White J. Goltzman D. Identification and characterization of 1,25-dihydroxyvitamin D3-responsive repressor sequences in the rat parathyroid hormone-related peptide gene.J Biol Chem. 1996; 271: 16310-16316Crossref PubMed Scopus (68) Google Scholar;Löwik et al., 1992Löwik Cwgm Hockman K. Offringa R. Groot C.G. Hendy G.N. Papapoulos S.E. Ponec M. Regulation of parathyroid hormonelike protein production in cultured normal and malignant keratinocytes.J Invest Derm. 1992; 98: 198-203Crossref PubMed Scopus (26) Google Scholar;Werkmeister et al., 1993Werkmeister J.R. Merryman J.I. McCauley L.K. Horton J.E. Capen C.C. Rosol T.J. Parathyroid hormone-related protein production by normal human keratinocytes in vitro.Exp Cell Res. 1993; 208: 68-74Crossref PubMed Scopus (34) Google Scholar;Falzon, 1996Falzon M. DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression.Mol Endo. 1996; 10: 672-681Crossref PubMed Scopus (76) Google Scholar). PTHrP has been variously reported to induce both increased and decreased proliferation of keratinocytesin vitro and there is also evidence to suggest that the peptide can directly affect keratinocyte differentiation (Henderson et al., 1992Henderson J.E. Kremer R. Rhim J.S. Goltzman D. Identification and functional characterization of adenylate cyclase-linked receptors for parathyroid hormone-like peptides on immortalized keratinocytes.Endocrinology. 1992; 130: 449-457Crossref PubMed Scopus (29) Google Scholar;Kaiser et al., 1991Kaiser S.T. Laneuville P. Bernier S.M. Rhim J.S. Kremer R. Goltzman D. Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.J Biol Chem. 1991; 267: 13623-13628Google Scholar,Kaiser et al., 1994Kaiser S.M. Sebag M. Rhim J.S. Kremer R. Goltzman D. Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation.Mol Endo. 1994; 8: 139-147Crossref PubMed Scopus (107) Google Scholar;Whitfield et al., 1996Whitfield J.F. Isaacs R.J. Jouishomme H. et al.C-terminal fragment of parathyroid hormone-related protein, PTHrP-(107–111), stimulates membrane-associated protein kinase C activity and modulates the proliferation of human and murine skin keratinocytes.J Cell Physiol. 1996; 166: 1-11Crossref PubMed Scopus (36) Google Scholar).In vivo, administration of PTH/PTHrP receptor agonists and antagonists in mice has been reported to induce inhibition and stimulation of epidermal proliferation, respectively (Holick et al., 1994Holick M.F. Ray S. Chen T.C. Persons KsA parathyroid hormone antagonist stimulates epidermal proliferation and hair growth in mice.Proc Natl Acad Sci USA. 1994; 91: 8014-8016Crossref PubMed Scopus (74) Google Scholar,Holick et al., 1996Holick M.F. Chen M.L. Kong X.F. Sanan D.K. Clinical uses for calciotropic hormones 1,25-dihydroxyvitamin D3 and parathyroid hormone-related peptide in dermatology: a new perspective.J Invest Derm Symp Proc. 1996; 1: 1-9PubMed Google Scholar;Schilli et al., 1997Schilli M.C. Ray S. Pau R. Obi-Tabot E. Holick M.F. Control of hair growth with parathyroid hormone (7–34).J Invest Derm. 1997; 108: 928-932Abstract Full Text PDF PubMed Scopus (43) Google Scholar). Over the past several years, a series of experiments with transgenic and knockout mice have implicated PTHrP as an attenuator of programmed differentiation in the hair follicle, mammary ductal epithelium, and endochondral bone. PTHrP overexpression in basal keratinocytes and outer root sheath cells as directed by the keratin-14 (K14) promoter caused either profound delay or frank failure in hair follicle initiation (Wysolmerski et al., 1994Wysolmerski J.J. Broadus A.E. Zhou J. Fuchs E. Milstone L.M. Philbrick W.M. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.Proc Natl Acad Sci USA. 1994; 91: 1133-1137Crossref PubMed Scopus (168) Google Scholar), and overexpression of the peptide in the mammary gland resulted in ductal hypoplasia (Wysolmerski et al., 1995Wysolmerski J.J. McCaughern-Carucci J.F. Daifotis A.G. Broadus A.E. Philbrick W.M. Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development.Development. 1995; 121: 3539-3547Crossref PubMed Google Scholar). Similarly, targeted overexpression of PTHrP to cartilage delayed both chondrocyte differentiation and endochondral ossification (Weir et al., 1996Weir E.C. Philbrick W.M. Amling M. Neff L.A. Baron R. Broadus A.E. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.Proc Natl Acad Sci USA. 1996; 93: 10240-10245Crossref PubMed Scopus (382) Google Scholar), whereas disruption of the PTHrP gene or its receptor resulted in a lethal skeletal dysplasia characterized by the premature maturation of chondrocytes and the accelerated mineralization of bone (Karaplis et al., 1994Karaplis A. Luz A. Glowacki J. Bronson R.J. Tybolewicz Vlj Kronenberg H.M. Mulligan R.C. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.Genes and Dev. 1994; 8: 277-289Crossref PubMed Scopus (959) Google Scholar;Lanske et al., 1996Lanske B. Karaplis A.C. Lee K. et al.PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth.Science (Wash DC). 1996; 273: 663-666Crossref PubMed Scopus (1073) Google Scholar). The early demise of the PTHrP-knockout animals, however, prevented analysis of the postnatal development of the skin and epidermal appendages. To circumvent this problem, we have taken advantage of a PTHrP-knockout mouse in which the lethal skeletal abnormalities have been corrected by the transgenic replacement of PTHrP in cartilage (Philbrick et al., 1998Philbrick W.M. Dreyer B.E. Nakchbandi I.A. Karaplis A.C. Parathyroid hormone-related protein is required for tooth eruption.Proc Natl Acad Sci USA. 1998; 95: 11846-11851Crossref PubMed Scopus (205) Google Scholar;Wysolmerski et al., 1998Wysolmerski J.J. Philbrick W.M. Dunbar M.E. Lanske B. Kronenberg H. Karaplis A. Broadus A.E. Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development.Development. 1998; 125: 1285-1294Crossref PubMed Google Scholar), and have found alterations in epidermal differentiation in the PTHrP-null state that appear to represent the converse of those seen in PTHrP overexpression. The generation of PTHrP-knockout mice, procollagen II-PTHrP transgenic mice, and K14-PTHrP transgenic mice, and the breeding involved in the generation of procollagen II (col II)-PTHrP/PTHrP-null mice and K14-PTHrP, col II-PTHrP/PTHrP-null mice has been described (Karaplis et al., 1994Karaplis A. Luz A. Glowacki J. Bronson R.J. Tybolewicz Vlj Kronenberg H.M. Mulligan R.C. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.Genes and Dev. 1994; 8: 277-289Crossref PubMed Scopus (959) Google Scholar;Wysolmerski et al., 1994Wysolmerski J.J. Broadus A.E. Zhou J. Fuchs E. Milstone L.M. Philbrick W.M. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.Proc Natl Acad Sci USA. 1994; 91: 1133-1137Crossref PubMed Scopus (168) Google Scholar,Wysolmerski et al., 1998Wysolmerski J.J. Philbrick W.M. Dunbar M.E. Lanske B. Kronenberg H. Karaplis A. Broadus A.E. Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development.Development. 1998; 125: 1285-1294Crossref PubMed Google Scholar;Philbrick et al., 1998Philbrick W.M. Dreyer B.E. Nakchbandi I.A. Karaplis A.C. Parathyroid hormone-related protein is required for tooth eruption.Proc Natl Acad Sci USA. 1998; 95: 11846-11851Crossref PubMed Scopus (205) Google Scholar;Weir et al., 1996Weir E.C. Philbrick W.M. Amling M. Neff L.A. Baron R. Broadus A.E. Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.Proc Natl Acad Sci USA. 1996; 93: 10240-10245Crossref PubMed Scopus (382) Google Scholar). Two independent lines of procollagen II-PTHrP transgenic and keratin 14-PTHrP transgenic mice were used in the crosses generating col II-PTHrP/PTHrP-null mice and K14-PTHrP, col II-PTHrP/PTHrP-null mice. In both cases, the independent transgenic lines produced identical skin phenotypes in the rescued PTHrP-knockout mice. At least two control littermates, rescued PTHrP-knockout, K14-PTHrP and doubly transgenic PTHrP-knockout mice, were analyzed for each time point. Skin samples (1–2 cm2) were removed from the mid-dorsum and abdomen of sacrificed mice and fixed directly in 10% buffered formalin (Baxter Chicago, IL), 70% ethanol or Bouin's fixative (Sigma, St. Louis, MO). Tissues were dehydrated, embedded in paraffin, and 5 μm sections were cut. Skin sections were visualized with routine hematoxylin and eosin staining. Skin samples from all rescued PTHrP-knockout mice were compared with control littermates that were one of the following genotypes: wild-type, col II-PTHrP transgenic, PTHrP ±, or col II-PTHrP/PTHrP ±. The skin samples from rescued PTHrP-knockout mice and their littermates were compared with age-matched K14-PTHrP transgenic mice. In preparation of the figures, all skin samples also were matched so that they represented similar points of the hair cycle. Because entrance into the second and third waves of anagen often occurs at different times among individuals of the same litter (Andreasen, 1953Andreasen E. Cyclic changes in the skin of the mouse.Acta Pathol Microbiol Immunol, Scand a. 1953; 32: 157-164Crossref Scopus (22) Google Scholar), occasionally skin samples from PTHrP-knockout mice samples were compared with controls from different litters and K14 samples derived from mice of slightly different ages. Sections were deparaffinized with xylene, hydrated via consecutive changes in decreasing concentrations of ethanol, and washed in dH2O. When required, antigen recovery was performed by pressure cooking sections for 1 min in 0.01 M citrate buffer pH 6.0. Sections were washed in Tris saline buffer (10 mM Tris-HCl, pH 7.4, 0.9% NaCl). Endogenous peroxidases were quenched for 20 min in methanol containing 0.5% H2O2. Slides were washed in Tris saline buffer and proteolytically processed for 5 min in 0.1% trypsin, 0.1% CaCl2 at pH 7.4–7.8. Sections were washed twice with Tris saline buffer, then bovine serum albumin buffer (20 mM Tris-HCl pH 8.2, 0.9% NaCl, 1% bovine serum albumin) and subsequently blocked in bovine serum albumin buffer containing 10% normal goat serum (Vector Laboratories, Burlingame, CA) for 20 min at room temperature. Primary antibodies were applied for 1 h at room temperature. Sections were washed as above and treated with biotinylated secondary antibodies, avidin peroxidase conjugates, and developed in the presence of DAB according to the manufacturer's instructions (Vector Laboratories). Sections were washed in dH2O, counterstained with methylene green (Sigma), and mounted with Permount (Fisher, Pittsburgh, PA). The following rabbit polyclonal antibodies were used at the listed concentrations and with the following alterations to the basic protocol: anti-mouse ki67 (Novocastra, Newcastle, U.K.) (1–1000; protease processing was omitted); anti-mouse keratin-6 and loricrin (Berkeley Antibody, Encino, CA) (1–3000), anti-mouse keratin-14 (gift from D. Roop, Baylor University, Houston, TX) (1–10,000; antigen recovery was omitted) and anti-mouse keratin 1 (gift from D. Roop, Baylor University) (1–5000). BrdU solution (Amersham, Arlington Heights, IL) was delivered to mice via intraperitoneal injection at 3 μg per gram body weight. Six hours post-injection mice were sacrificed and tissues harvested and placed in Bouin's fixative. Skin sections were then embedded and prepared using standard techniques. BrdU incorporation was evaluated using an anti-BrdU monoclonal antibody and labeling kit (Cell Proliferation Kit, Amersham) according to the manufacturer's instructions. The relative proliferation index was determined by calculating the percentage of labeled nuclei per 200 basal nuclei. In the case of the K14-PTHrP ventral epidermis, nuclei from the first two layers were considered to be basal nuclei. Ten sets of 200 cells were counted for each skin sample, and the number of labeled cells from each of these sets were averaged. The perinatal death of the PTHrP-knockout mice appears to be due to inappropriate ossification of the costal cartilage and subsequent respiratory failure (Karaplis et al., 1994Karaplis A. Luz A. Glowacki J. Bronson R.J. Tybolewicz Vlj Kronenberg H.M. Mulligan R.C. Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.Genes and Dev. 1994; 8: 277-289Crossref PubMed Scopus (959) Google Scholar). Specific replacement of PTHrP expression in the chondrocytes of knockout mice with a procollagen II-PTHrP transgene has recently been shown to produce a viable animal that is effectively PTHrP-null in all tissues except cartilage (Philbrick et al., 1998Philbrick W.M. Dreyer B.E. Nakchbandi I.A. Karaplis A.C. Parathyroid hormone-related protein is required for tooth eruption.Proc Natl Acad Sci USA. 1998; 95: 11846-11851Crossref PubMed Scopus (205) Google Scholar;Wysolmerski et al., 1998Wysolmerski J.J. Philbrick W.M. Dunbar M.E. Lanske B. Kronenberg H. Karaplis A. Broadus A.E. Rescue of the parathyroid hormone-related protein knockout mouse demonstrates that parathyroid hormone-related protein is essential for mammary gland development.Development. 1998; 125: 1285-1294Crossref PubMed Google Scholar). These rescued PTHrP-knockout mice display a number of developmental defects in ectodermally derived structures, including a failure of mammary branching morphogenesis and the absence of tooth eruption. These animals also exhibit abnormalities in the skin and related appendages, including a matted, oily coat (Figure 1a), thinning of the coat with age (Figure 1c), long, malformed nails (Figure 1a,d), and scaling and flaking of the foot pads (Figure 1d). Some scaling and flaking was also noted in both ventral and dorsal skin following clipping of the hair (not shown). Multiple histologic abnormalities were apparent in the skin of rescued PTHrP-knockout mice (Figure 1c,d). The epidermis from both the dorsal and the ventral skin was reduced in thickness and was composed of an eosinophilic basal layer of cells that contained condensed nuclei and displayed a squamous morphology. The suprabasal layers were extremely thin, and the outer cornified layer exhibited marked hyperkeratosis. The sebaceous glands appeared hypoplastic and contained decreased numbers of sebocytes. The dermis from these animals appeared fibrotic and contained numerous eosinophilic collagen fibrils and there was a marked reduction in dermal fat. Also, although dorsal skin is easily distinguished from ventral skin in wild-type animals by the presence of a thicker dermal fat layer, smaller sebaceous glands, larger hair follicles, and an epidermis with more regular invaginations (Figure 2a,b), such morphologic differences were much less obvious in the rescued PTHrP-knockout mice. Overexpression of PTHrP in the skin with the human keratin-14 promoter has previously been shown to result in a reduction in the density of hair follicles (Wysolmerski et al., 1994Wysolmerski J.J. Broadus A.E. Zhou J. Fuchs E. Milstone L.M. Philbrick W.M. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.Proc Natl Acad Sci USA. 1994; 91: 1133-1137Crossref PubMed Scopus (168) Google Scholar). This was most striking in the ventral skin, where expression of the transgene was highest and hair follicles were essentially absent. As shown in (Figure 2e), the histologic changes in the dorsal skin of the K14-PTHrP transgenic mice were fairly mild, consisting of a slight increase in the cellularity of the epidermis and thickening of the dermal collagen layer. The ventral interfollicular epidermis (Figure 2f), however, displayed a densely packed basal layer of elongated, columnar keratinocytes, an increased number of suprabasal layers, which included cells that retained nuclei, and a mildly hyperkeratotic cornified layer. Sebaceous glands were reduced in number, but they appeared hyperplastic and contained increased numbers of sebocytes within the glands. The ventral dermis from K14-PTHrP mice appeared to be very cellular, collagen fibers were less tightly packed than in control animals, and there was a reduction in dermal fat. These changes are at their most extreme in the ventral surface of female K14-PTHrP transgenic mice, which exhibited both acanthosis and a dramatic papillomatosis. The epidermal alterations in male mice are mitigated somewhat, most likely due to the influence of androgens (Wysolmerski et al., 1994Wysolmerski J.J. Broadus A.E. Zhou J. Fuchs E. Milstone L.M. Philbrick W.M. Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.Proc Natl Acad Sci USA. 1994; 91: 1133-1137Crossref PubMed Scopus (168) Google Scholar). Histologic findings in the epidermis of the footpads and tails of K14-PTHrP and rescued PTHrP-knockout mice paralleled the respective findings in the ventral skin of these animals. Thus, in many respects, the histologic changes observed in the interfollicular epidermis and the sebaceous glands of the rescued PTHrP-knockout and K14-PTHrP transgenic mice appear to be mirror images of one another, reinforcing the notion that PTHrP functions as an attenuator of epidermal and adnexal proliferation and/or differentiation. The effects of PTHrP overexpression can first be detected at embryonic day 18 (E18), as a delay in the initiation of dorsal hair follicles. To establish the time course of the pathologic changes observed in the PTHrP-null state, a series of skin samples were taken from PTHrP-knockout mice at 18 d post-coitum and birth, and from rescued PTHrP-knockout mice at 1, 2, 3, 4, 5, 6, 8, 12, 16, and 20 wk of age (rescued PTHrP-knockout mice generally do not live beyond 24 wk). No significant histologic changes were found in the skin of E18 or newborn PTHrP-knockout mice or in 1 wk old rescued PTHrP-knockout mice (Figure 3a,b). At 3–4 wk of age, however, a histopathologic pattern became apparent that closely resembled that observed in adult PTHrP-null skin and included thinning of the epidermis, hyperkeratosis, hypoplastic sebaceous glands, and alterations in collagen fibers (Figure 3c,d). These changes became progressively more striking in older (12–20 wk) rescued PTHrP-knockout mice in which the epidermis had become extremely thin and was covered by multiple layers of hyperkeratotic material (Figure 3e,f). Thus, PTHrP does not appear to be essential for the early morphogenesis of the epidermis and dermis, but rather appears to be required to maintain proper skin development with increasing age. The contrasting histologic findings seen in states of PTHrP overexpression and PTHrP underexpression suggested that this peptide affects either the proliferation or the differentiation of keratinocytes. Only the basal keratinocytes within the murine epidermis have proliferative capacity and this proliferation is normally under tight control (Ebling et al., 1992Ebling Fjg Eady Raj Leigh I.M. Anatomy and organization of human skin.Textbook of Dermatology. London, Blackwell Scientific Publications1992: 49-124Google Scholar). By inspection, the ventral skin of the K14-PTHrP mice appeared to have increased numbers of epidermal keratinocytes as compared with controls and this increased cellularity involved both the basal and the suprabasal layers (Figure 2f). In the adult rescued PTHrP-knockout mice, cell counts of defined lengths of ventral epidermis indicated that there was a 20% decrease in the number of nuclei in the basal layer (≈800 per cm) as compared with similar counts with controls (≈1000 per cm). This type of analysis was not possible, however, in K14-PTHrP mice due to the acutely furrowed ventral epidermis in these animals. Therefore, to directly evaluate relative proliferation rates in the epidermis from rescued PTHrP-knockout, K14-PTHrP-transgenic and control animals, BrdU was employed to label cells in S-phase. As anticipated, BrdU incorporation was largely confined to the basal layer in the epidermis in all three animals (not shown). A relative proliferation index was generated by counting the number of labeled cells per 2000 basal cell nuclei in sections from each of the mice. Approximately 2.0% of the basal cells were labeled in both control and rescued PTHrP-knockout epidermis (Figure 4) and this was increased to 5.7% in K14-PTHrP mice. Similar results were obtained using an antibody to the ki67 epitope expressed by cells in the late G1, S, G2, and M phases of the cell cycle (Key et al., 1993Key G. Becker M.H. Baron B. Duchrow M. Schluter C. Flad H.D. Gerdes J. New Ki-67-equivalent murine monoclonal antibodies (MIB 1–3) against bacterially expressed parts of the Ki-67 cDNA containing three base pair repetitive elements encoding for the Ki-67 epitope.Lab Invest. 1993; 68: 629-636PubMed Google Scholar) (data not shown). Thus, although the absence of PTHrP has no apparent effect on epidermal proliferation, overexpression of the peptide results in a modest increase in the proliferation rate of basal keratinocytes. Under conditions such as wound healing, psoriasis, and various neoplastic states, keratinocytes can assume a hyperproliferative phenotype in which the epidermis becomes thickened, cell division occurs in suprabasal layers and a unique set of differentiation markers, including keratin 6 (K6), are expressed (Schweizer, 1993Schweizer J. Murine epidermal keratins.in: Bloomberg M. Molecular Biology of the Skin. The Keratinocyte. Academic Press, New York1993: 33-78Google Scholar;Heyden et al., 1994Heyden A. Lützow-Holm C. Clausen Opf et al.Application of cantharidin or 12–0-tetradecanoylphorbol-13-acetate on mouse epidermis induces a cell population shift that causes altered keratin distribution.Differentiation. 1994; 57: 187-193Crossref Pu