A Novel Keratinocyte Mitogen: Regulation of Leptin and its Functional Receptor in Skin Repair
2001; Elsevier BV; Volume: 117; Issue: 1 Linguagem: Inglês
10.1046/j.0022-202x.2001.01387.x
ISSN1523-1747
AutoresBirgit Stallmeyer, Heiko Kämpfer, Josef Pfeilschifter, Stefan L. Frank, Maurizio Podda, Roland Kaufmann,
Tópico(s)melanin and skin pigmentation
ResumoWound re-epithelialization represents a tissue movement that crucially participates in wound closure. Recently, we demonstrated that supplemented leptin improved re-epithelialization processes in leptin-deficient ob/ob mice. In this study we investigated regulation of the leptin system during normal repair in healthy animals. We found leptin to be present at the wound site during healing, although leptin levels were clearly reduced upon injury compared with uninvolved control skin. The functional leptin receptor subtype obRb was observed to be constitutively expressed in nonwounded skin. During early healing, the leptin receptor obRb was downregulated, but re-increased again from day 5 postwounding. Immunohistochemistry revealed that highly proliferative keratinocytes of the wound margin epithelia strongly expressed the functional leptin receptor subtype obRb. In vitro studies demonstrated that murine and human primary epidermal keratinocytes responded to exogenously added leptin with a proliferative response. Moreover, specificity of leptin-mediated mitogenic effects on primary keratinocytes could be shown by completely blocking leptin actions by a soluble, nonfunctional chimeric leptin receptor. Finally, we report that leptin, besides the recently described activation of the janus tyrosine kinase signal transducers, also activated extracellular signal-regulated kinase-controlled signaling pathways in primary keratinocytes. Wound re-epithelialization represents a tissue movement that crucially participates in wound closure. Recently, we demonstrated that supplemented leptin improved re-epithelialization processes in leptin-deficient ob/ob mice. In this study we investigated regulation of the leptin system during normal repair in healthy animals. We found leptin to be present at the wound site during healing, although leptin levels were clearly reduced upon injury compared with uninvolved control skin. The functional leptin receptor subtype obRb was observed to be constitutively expressed in nonwounded skin. During early healing, the leptin receptor obRb was downregulated, but re-increased again from day 5 postwounding. Immunohistochemistry revealed that highly proliferative keratinocytes of the wound margin epithelia strongly expressed the functional leptin receptor subtype obRb. In vitro studies demonstrated that murine and human primary epidermal keratinocytes responded to exogenously added leptin with a proliferative response. Moreover, specificity of leptin-mediated mitogenic effects on primary keratinocytes could be shown by completely blocking leptin actions by a soluble, nonfunctional chimeric leptin receptor. Finally, we report that leptin, besides the recently described activation of the janus tyrosine kinase signal transducers, also activated extracellular signal-regulated kinase-controlled signaling pathways in primary keratinocytes. Wound healing disorders are a therapeutic problem of extensive clinical importance. Thus, it is reasonable to search for novel factors regulating wound healing processes to establish novel therapeutic approaches. Re-epithelialization is central to wound closure and driven by movements of epithelial keratinocytes. After a lag period of several hours after skin injury, these movements are initiated starting with epidermal migration (Stenn et al., 1988Stenn K.S. Depalma L. Re-epithelialization.in: Clark R.A.F. Hensen P.M. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1988: 321-325Crossref Google Scholar;Martin, 1997Martin P. Wound healing – aiming for perfect skin regeneration.Science. 1997; 276: 75-81https://doi.org/10.1126/science.276.5309.75Crossref PubMed Scopus (3430) Google Scholar). Subsequently, the injury-mediated loss of epidermal keratinocytes is replaced by the generation of keratinocytes from the newly formed highly proliferative epithelia at the wound margins, which now feed the moving epithelial tongue. Growth factors and cytokines participate in the close regulation of keratinocyte behavior during these processes. Thus, epidermal growth factor (EGF) and keratinocyte growth factor (KGF) are well known to be potent mitogens for epithelial cells, and therefore essentially involved in re-epithelialization during skin repair (Nanney, 1990Nanney L.B. Epidermal and dermal effects of epidermal growth factor and its receptors during wound repair.J Invest Dermatol. 1990; 94: 624-629Abstract Full Text PDF PubMed Google Scholar;Werner et al., 1994Werner S. Smola H. Liao X. Longaker M.T. Krieg T. Hofschneider P.H. Williams L.T. The function of KGF in morphogenesis of epithelium and reepithelialization of wounds.Science. 1994; 266: 819-822Crossref PubMed Scopus (490) Google Scholar;Marchese et al., 1995Marchese C. Chedid M. Dirsch O.R. et al.Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.J Exp Med. 1995; 182: 1369-1376Crossref PubMed Scopus (142) Google Scholar). Recently, we revealed that the ob gene product leptin, which has been characterized as a satiety-regulating cytokine and which is predominantly expressed by adipocytes and secreted into the bloodstream (Zhang et al., 1994Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11221) Google Scholar;Masuzaki et al., 1995Masuzaki H. Ogawa Y. Isse N. et al.Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue.Diabetes. 1995; 44: 855-858Crossref PubMed Scopus (0) Google Scholar), markedly improves the severely disturbed wound healing conditions that are associated with the loss of a functional leptin system in obese/obese (ob/ob) mice (Frank et al., 2000Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (235) Google Scholar). Systemically and also topically administered recombinant leptin improved re-epithelialization of excisional wounds in the leptin-deficient ob/ob mouse model of impaired skin repair. Leptin is structurally related to cytokines (Zhang et al., 1997Zhang F. Basinski M.B. Beals J.M. et al.Crystal structure of the obese protein leptin-E100.Nature. 1997; 387: 206-209Crossref PubMed Scopus (539) Google Scholar) and acts on receptors that have considerable homology to class I cytokine receptors (Tartaglia et al., 1995Tartaglia L.A. Dembski M. Weng X. et al.Identification and expression cloning of a leptin receptor, OB-R.Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3127) Google Scholar). Among several leptin receptor (obR) isoforms, the appetite-regulating effect of leptin has been shown to be dependent on binding of leptin to the corresponding leptin receptor subtype obRb in the hypothalamic region (Ghilardi et al., 1996Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Defective STAT signaling by the leptin receptor in diabetic mice.Proc Natl Acad Sci USA. 1996; 93: 6231-6235https://doi.org/10.1073/pnas.93.13.6231Crossref PubMed Scopus (719) Google Scholar;Lee et al., 1996Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Abnormal splicing of the leptin receptor in diabetic mice.Nature. 1996; 379: 632-635Crossref PubMed Scopus (2060) Google Scholar;Vaisse et al., 1996Vaisse C. Halaas J.L. Horvath C.M. Darnell Jr, Je Stoffel M. Friedman J.M. Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice.Nat Genet. 1996; 14: 95-97Crossref PubMed Scopus (915) Google Scholar). Leptin function is restricted to binding of its only currently known functional receptor variant obRb. The obRb splice variant is the only leptin receptor that mediates an intracellular signaling by transphosphorylation of janus tyrosine kinases (jak) followed by a subsequent activation of signal transducers and activators of transcription (STAT) proteins (Ihle, 1995Ihle J.N. Cytokine receptor signalling.Nature. 1995; 377: 591-594Crossref PubMed Scopus (1111) Google Scholar;Bjorbaek et al., 1997Bjorbaek C. Uotani S. da Silva B. Flier J.S. Divergent signaling capacities of the long and short isoforms of the leptin receptor.J Biol Chem. 1997; 272: 32686-32695https://doi.org/10.1074/jbc.272.51.32686Crossref PubMed Scopus (757) Google Scholar). The absence of a functional leptin in ob/ob mice (Zhang et al., 1994Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11221) Google Scholar) or, additionally, the loss of the intracellular domain of the leptin receptor subtype obRb in db/db mice (Chen et al., 1996Chen H. Charlat O. Tartaglia L.A. et al.Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice.Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1859) Google Scholar;Lee et al., 1996Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Abnormal splicing of the leptin receptor in diabetic mice.Nature. 1996; 379: 632-635Crossref PubMed Scopus (2060) Google Scholar) results in decreased lipolytic activity and an increase of food intake (Halaas et al., 1995Halaas J.L. Gajiwala K.S. Maffei M. et al.Weight-reducing effects of the plasma protein encoded by the obese gene.Science. 1995; 269: 543-546Crossref PubMed Scopus (4103) Google Scholar;Ghilardi et al., 1996Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Defective STAT signaling by the leptin receptor in diabetic mice.Proc Natl Acad Sci USA. 1996; 93: 6231-6235https://doi.org/10.1073/pnas.93.13.6231Crossref PubMed Scopus (719) Google Scholar;Vaisse et al., 1996Vaisse C. Halaas J.L. Horvath C.M. Darnell Jr, Je Stoffel M. Friedman J.M. Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice.Nat Genet. 1996; 14: 95-97Crossref PubMed Scopus (915) Google Scholar). Thus, these animals exhibit a severe obesity and a diabetic phenotype (Coleman, 1982Coleman D.L. Diabetes obesity syndromes in mice.Diabetes. 1982; 31: 1-6Crossref PubMed Google Scholar). Although supplemented leptin has been recently described to reconstitute epithelial regeneration defects in leptin-deficient ob/ob mice and wild-type mice (Frank et al., 2000Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (235) Google Scholar), the regulation and potential function of the leptin-obRb leptin receptor system remains to be investigated, especially in nondisturbed wound healing processes in healthy animals. To this end, we determined expression patterns of leptin and its receptor subtype obRb during normal skin repair. Here we demonstrate the presence of leptin at the wound site and a transient decline in obRb expression levels upon injury. Moreover, especially proliferating keratinocytes located at the wound margins strongly expressed the functional leptin receptor obRb. These in vivo findings were further supported by the observation that leptin mediated a mitogenic stimulus to primary keratinocytes in vitro. Thus, our data suggest a novel role for leptin as a keratinocyte mitogen during skin repair. Female BALB/C mice were obtained from Charles River (Sulzfeld, Germany) and maintained under a 12 h light/12 h dark cycle at 22°C until they were 12 wk of age. At this time they were caged by four, monitored for body weight, and wounded as described below. To examine leptin functions on the wound healing process, six full-thickness wounds were created on the backs of female BALB/C mice. Animals were anesthetized with a single intraperitoneal injection of Ketamin (80 mg per kg body weight)/Xylazin (10 mg per kg body weight). The hair on the back of the mice was cut, and the back was subsequently wiped with 70% ethanol. Six full-thickness wounds (5 mm in diameter, 3–4 mm apart) were made on the backs of the mice by excising the skin and the underlying panniculus carnosus. The wounds were allowed to form a scab. Skin biopsy specimens from four animals were obtained 6 h, or 1, 3, 5, 7, and 13 d after injury. An area 7–8 mm in diameter that included the scab and the complete epithelial margins was excised at each time point. As controls, a similar amount of nonwounded skin was taken from the backs of four nonwounded mice, and from the backs of wounded animals, respectively. For every experimental time point, the wounds from four animals and the nonwounded back skin from four animals were combined and used for RNA (n = 16 wounds) and protein isolation (n = 8 wounds), respectively. All animal experiments were carried out according to the guidelines and with the permission of the local government of Hessen. RNA isolation was performed as described previously (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62307) Google Scholar). Twenty micrograms of total RNA from wounded or nonwounded skin were used for RNase protection assays. RNase protection assays were carried out as described previously (Werner et al., 1992Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M.J. Williams L.T. Large induction of keratinocyte growth factor expression in the dermis during wound healing.Proc Natl Acad Sci USA. 1992; 89: 6896-6900Crossref PubMed Scopus (514) Google Scholar), with four different sets of RNA from four independent wound healing experiments. The murine cDNA probes for leptin and the leptin receptor obRb were cloned by reverse-transcriptase polymerase chain reaction (RT-PCR). The cloned cDNA fragments correspond to nucleotides 62–389 (for leptin) or nucleotides 506–871 (for leptin receptor obR) of the published sequences (Zhang et al., 1994Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11221) Google Scholar;Tartaglia et al., 1995Tartaglia L.A. Dembski M. Weng X. et al.Identification and expression cloning of a leptin receptor, OB-R.Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3127) Google Scholar). RNases A and T1 were from Roche Biochemicals (Mannheim, Germany). Skin lysates were prepared as described previously (Frank et al., 1999Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (204) Google Scholar;Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098https://doi.org/10.1046/j.1523-1747.1999.00784.xAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Total protein (50 µg) from nonwounded back skin and 1, 3, 5, 7, and 13 d wounds were separated using sodium dodecyl sulfate gel electrophoresis. For the cell culture experiments, cell lysates from murine or human primary keratinocytes were prepared as described previously (Kämpfer et al., 1999Kämpfer H. Kalina U. Mühl H. Pfeilschifter J. Frank S. Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.J Invest Dermatol. 1999; 113: 369-374https://doi.org/10.1046/j.1523-1747.1999.00704.xAbstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). After transfer to a PVDF membrane, leptin receptor obRa- and obRb-specific proteins were detected using a polyclonal antiserum directed against a synthetic peptide whose sequence is derived from amino acids 577–594 of the mouse leptin receptor (ABR, Golden, CO). Phosphorylated extracellular-factor-regulated kinase 1 (pERK 1) -specific or ERK-1-specific proteins were determined using polyclonal antibodies raised against the rat ERK-1-encoded p44 (for pERK 1) or human ERK 1 (for ERK 1) (Santa Cruz, Heidelberg, Germany), respectively. A secondary antibody coupled to horseradish peroxidase (Biomol, Hamburg, Germany) and the enhanced chemiluminescence detection system were used to visualize leptin receptor protein. Mice were wounded as described above. At days 5, 7, and 13 after wounding mice were sacrificed, and complete wounds were isolated from the middle of the back, bisected, and frozen in tissue-freezing medium. Immunohistochemistry was carried out on directly neighboring 6 µm frozen serial sections as described previously (Frank et al., 1999Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.FASEB J. 1999; 13: 2002-2014Crossref PubMed Scopus (204) Google Scholar;Stallmeyer et al., 1999Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Frank S. The function of nitric oxide in wound repair: inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization.J Invest Dermatol. 1999; 113: 1090-1098https://doi.org/10.1046/j.1523-1747.1999.00784.xAbstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Sections were incubated for 60 min at room temperature with a polyclonal antibody raised against the COOH-terminus of the murine obRb leptin receptor subtype (Santa Cruz, Heidelberg, Germany), or a polyclonal antiserum raised against murine Ki67 (Dianova, Hamburg, Germany) diluted 1:100 in phosphate-buffered saline, 0.1% bovine serum albumin. Murine primary epidermal keratinocytes were isolated and maintained as described byHager et al., 1999Hager S. Bickenbach J.R. Fleckman P. Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976https://doi.org/10.1046/j.1523-1747.1999.00605.xCrossref PubMed Scopus (105) Google Scholar in a defined keratinocyte medium (KBM) containing 0.06 mM Ca2+ (BioWhittaker Europe, Verviers, Belgium). Human primary epidermal keratinocytes were purchased from BioWhittaker Europe. Cells were cultured in a defined keratinocyte medium (KBM-2) according to the instructions of the supplier. Murine primary keratinocytes were seeded on 96-well plates. Each well contained 103 cells in a total volume of 100 µl of KBM. Subsequently, cells were grown for 24 h. The proliferation rate of incubated cells was determined after a 24 h stimulation with leptin (100 ng per ml) using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Mannheim, Germany) according to the instructions of the manufacturer. This assay determines the number of viable cells in proliferation assays, because the total amount of the formed formazan end product is directly proportional to the number of living cells in culture. For the experiment using the nonfunctional leptin receptor, 5 × 102 cells were seeded and leptin-stimulated (1 ng per ml) for 48 h in the presence or absence of the leptin receptor/IgG1 Fc chimeric protein (100 ng per ml) (R&D Systems, Wiesbaden, Germany) before proliferation rates were determined. Murine recombinant leptin was purchased from R&D Systems, and KGF and EGF were from Roche Biochemicals. Human primary keratinocytes were seeded on 96-well plates. Each well contained 5 × 102 cells in a total volume of 100 µl of KBM-2. Subsequently, cells were grown for 24 h. The proliferation rate of incubated cells was determined after a 24 h stimulation with leptin (100 ng per ml), KGF (10 ng per ml), or EGF (10 ng per ml). After growth factor incubation, 5-bromo-2′-deoxyuridine (BrdU) labeling reagent (Roche Biochemicals) was added for an additional 1 h incubation. BrdU incorporation during cellular S-phase, as assessed by BrdU colorimetric cell proliferation enzyme-linked immunosorbent assay (ELISA) (Roche Biochemicals), was used as a direct readout for cell proliferation rates. Human recombinant leptin was from R&D Systems. Total protein (50 µg diluted in lysis buffer to a final volume of 50 µl) from nonwounded skin lysates and wound lysates, respectively, was subsequently analyzed for the presence of immunoreactive leptin protein by ELISA using the Quantikine murine leptin kit (R&D Systems) as described by the manufacturer. Data are shown as means ± SD. Data were analyzed by unpaired Student's t test on raw data using Sigma Plot (Jandel Scientific, Erkrath, Germany). As we have shown leptin to enhance wound closure in leptin-deficient ob/ob mice (Frank et al., 2000Frank S. Stallmeyer B. Kämpfer H. Kolb N. Pfeilschifter J. Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.J Clin Invest. 2000; 106: 501-509Crossref PubMed Scopus (235) Google Scholar), we were interested in whether the leptin-obRb leptin receptor system might also participate in normal repair. Therefore we first investigated the time course of leptin mRNA and protein expression during this process in healthy BALB/C mice. We isolated total RNA from full-thickness excisional wounds at different intervals after injury and performed RNase protection assays. RNA from four independent wound healing experiments was analyzed for leptin expression. For the RNase protection assay shown in Figure 1(a) (upper panel), it should be noted that every experimental time point represents 16 control skin or wound biopsy specimens (n = 16) isolated from four animals. As shown in Figure 1, we could detect a low level of basal expression of leptin mRNA in normal back skin (ctrl skin). Upon injury, a rapid increase (about 3–4-fold) in leptin mRNA expression was observed. The observed increase was only transient, as leptin mRNA levels were only slightly elevated from day 3 postwounding. It should be noted that every experimental time point in Figure 1(a) (lower panel) represents a total of 64 control skin or wound biopsy specimens (n = 64) from four independent wound healing experiments. As a next step, we determined whether the increase in leptin mRNA levels upon injury was subsequently followed by increasing leptin protein levels within the wound. This was important, as leptin is mainly produced by adipocytes and secreted into the bloodstream (Zhang et al., 1994Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Positional cloning of the mouse obese gene and its human homologue.Nature. 1994; 372: 425-432Crossref PubMed Scopus (11221) Google Scholar;Masuzaki et al., 1995Masuzaki H. Ogawa Y. Isse N. et al.Human obese gene expression. Adipocyte-specific expression and regional differences in the adipose tissue.Diabetes. 1995; 44: 855-858Crossref PubMed Scopus (0) Google Scholar). This observation raised the question whether locally expressed leptin mRNA at the wound site Figure 1a translates into leptin protein that is available at the site of repair rather than "being lost" from the wound site by secretion into the blood stream. Furthermore, leptin that was not produced in the skin but in other organ systems of the body might also be transported into the wound via the vasculature. To clarify this question, we measured leptin concentrations in normal skin and wound lysates using a sensitive ELISA technique. We observed a constitutive expression of leptin in nonwounded back skin, which markedly declined upon injury by about 50% Figure 1b. The decline in leptin protein was only transient, as we determined re-increasing leptin levels from day 3 postwounding that nearly reached levels of nonwounded skin after 13 d of repair. As we had observed leptin to be present at the wound site, we subsequently investigated the expression pattern of the leptin receptor in normal skin and during cutaneous repair. Until now, six different splice variants of the leptin receptor gene have been identified, of which only the obRb isoform has been demonstrated to mediate intracellular signaling (Ghilardi et al., 1996Ghilardi N. Ziegler S. Wiestner A. Stoffel R. Heim M.H. Skoda R.C. Defective STAT signaling by the leptin receptor in diabetic mice.Proc Natl Acad Sci USA. 1996; 93: 6231-6235https://doi.org/10.1073/pnas.93.13.6231Crossref PubMed Scopus (719) Google Scholar). To investigate regulation of obR mRNA and protein expression during repair, we performed RNase protection assay Figure 2a, Western blot analysis Figure 2b, and immunohistochemistry Figure 4. We used an antisense RNA probe that hybridized to an mRNA region recognizing both obRa and obRb. As shown in Figure 2, obR mRNA and protein were expressed in nonwounded skin, but wounding led to a rapid decline in obR mRNA and protein levels. Minimal obR expression was observed 3 d after wounding. obR mRNA levels started to re-increase again 5 d after injury Figure 2a. Weak signals for obRb-specific proteins could be detected after 7 d postwounding, but clearly visible signals appeared after 13 d of repair Figure 2b. For the wound healing experiments, skin from nonwounded mice served as the control that represented the normal, noninjured situation. Nevertheless, it is tempting to argue that the trauma of wounding, including anesthesia, pain, or systemic effects resulting from surgery, might alter basal leptin and leptin receptor (obR) expression. Thus, possible alterations in leptin and obR expression upon wounding surgery might contribute to the observed expressional changes in leptin and its receptor at the wound site. To exclude systemic effects of the wounding procedure on leptin and obR expression, mice were wounded as described (see Materials and Methods). Six hours or 24 h after the initial wounding, respectively, nonwounded skin located adjacent to the wound sites was taken as an additional control. As shown in Figure 3, wounding did not significantly alter cutaneous expression of leptin Figure 3a or leptin receptor Figure 3b mRNA in nonwounded skin areas that now served as additional controls. Accordingly, expression of leptin and its receptor obR was not influenced by systemic effects induced by the wounding procedure. The ctrl skin lane represents a total of 16 biopsy specimens of nonwounded skin (n = 16) from four different animals (n = 4) that had been pooled prior to analysis. It is important to note that the other data points depicted represent three biopsy specimens each of nonwounded skin (n = 3) that had been isolated for analysis from "control-wounded" individual animals (mouse #1 to #6) 6 h and 24 h after injury. The absence of obRb-specific signals at days 5–7 after injury, compared to re-increasing obR mRNA expression at these time points, could be due to a limited sensitivity of the Western blot analysis, especially as many cytokine receptors are known to be expressed in limited amounts. To solve this problem, we performed a sensitive immunohistochemistry analysis using 6 µm frozen serial sections from 5, 7, and 13 d wounds. Furthermore, as leptin was available at the wound site, it was important to determine the potential target cells for leptin at the wound site. As shown in Figure 4, we indeed detected the obRb receptor variant to be strongly expressed in 5, 7, and 13 d wounds, thus confirming the data obtained at the mRNA level. In control skin, obRb protein was specifically expressed in keratinocytes of the basal layer of the epidermis (data not shown). The same expression pattern for obRb was observed in completely re-epithelialized wounds 13 d after injury (Figure 4, lower panel). Moreover, obRb expression was not restricted to the basal layer of the epidermis during repair, as strong immunopositive signals could be detected in keratinocytes of the hyperproliferative epithelia at the wound margins (Figure 4, upper panel). As expression of the obRb leptin receptor isoform was clearly confined to proliferating keratinocytes of the epidermal basal layer and the hyperproliferative epithelium, we hypothesized that leptin might mediate a mitogenic stimulus to keratinocytes located at the wound margins. To this end, we stained directly neighboring 6 µm serial frozen sections from 5, 7, and 13 d wounds for obRb protein expression and expression of the proliferation marker Ki67 (Gerdes et al., 1984Gerdes J. Lemke H. Baisch H. Wacker H.H. Schwab U. Stein H. Cell cycle analysis of a cell proliferation associated human nuclear antigen defined by the monoclonal antibody Ki67.J Immunol. 1984; 133: 1709-1715Google Scholar), respectively Figure 4. Not unexpectedly, we observed a clear correlation between obRb-immunopositive (Figure 4, left panels) and proliferating (Figure 4, right panels) keratinocytes. It is noteworthy that the obRb was expressed throughout nearly all keratinocytes of the hyperproliferative epithelia (Figure 4, upper left panel). This phenomenon clearly correlated with the highly mitogenic activity of wound margin keratinocytes at this stage of tissue regeneration (Figure 4, upper right panel), whereas keratinocyte obRb expression and, additionally, keratinocyte mitogenic activity became more and more restricted to the basal layers of the forming neo-epithelium at later stages of repair (Figure 4, middle and lower panels). To further strengthen our in vivo observations, we determined the mitogenic potency of leptin in murine and human primary keratinocytes in vitro. First, we investigated whether isolated murine and human primary keratinocytes expressed the obRb receptor subtype and thus could respond as target cells for leptin functions. Indeed, we could assess the presence of the
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