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Induction of Bone Morphogenetic Protein-6 in Skin Wounds. Delayed Reepitheliazation and Scar Formation in BMP-6 Overexpressing Transgenic Mice

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

10.1046/j.1523-1747.1998.00407.x

1523-1747

Sibylle Kaiser, Peter Schirmacher, Armin Philipp, Martina Protschka, Ingrid Moll, Karin Nicol, Manfred Blessing,

Surgical Sutures and Adhesives

Growth factors of the transforming growth factor-β superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-β related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cells in the wound bed. BMP-6-specific RNA was induced within 24 h after injury, whereas significant upregulation of BMP-6 on the protein level was detected only 2–3 d after injury. Protein was confined to outermost suprabasal epidermal layers, whereas BMP-6-specific RNA was distributed throughout all epidermal layers including basal keratinocytes and the leading edge of the migrating keratinocytes. We also detected high levels of BMP-6-specific RNA and protein in chronic human wounds of different etiology. In contrast to the overall distribution pattern of BMP-6-specific RNA, the protein was not detected in keratinocytes directly bordering the wound. In order to test the influence of BMP-6 abundance on the progress of wound healing, we analyzed the wound response of transgenic mice overexpressing BMP-6 in the epidermis. In these mice, reepitheliazation of skin wounds was significantly delayed, suggesting that strict spatial and temporal regulation of BMP-6 expression is necessary not only for formation but also for reestablishment of a fully differentiated epidermis. Growth factors of the transforming growth factor-β superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-β related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cells in the wound bed. BMP-6-specific RNA was induced within 24 h after injury, whereas significant upregulation of BMP-6 on the protein level was detected only 2–3 d after injury. Protein was confined to outermost suprabasal epidermal layers, whereas BMP-6-specific RNA was distributed throughout all epidermal layers including basal keratinocytes and the leading edge of the migrating keratinocytes. We also detected high levels of BMP-6-specific RNA and protein in chronic human wounds of different etiology. In contrast to the overall distribution pattern of BMP-6-specific RNA, the protein was not detected in keratinocytes directly bordering the wound. In order to test the influence of BMP-6 abundance on the progress of wound healing, we analyzed the wound response of transgenic mice overexpressing BMP-6 in the epidermis. In these mice, reepitheliazation of skin wounds was significantly delayed, suggesting that strict spatial and temporal regulation of BMP-6 expression is necessary not only for formation but also for reestablishment of a fully differentiated epidermis. Cutaneous injury gives rise to a cascade of signals that start and control a series of processes required to reinstall the epidermal barrier. Inflammation, formation of granulation tissue, reepitheliazation, and matrix remodeling require the corroborative activity of distinct epidermal and dermal cell populations (Clark, 1985Clark R.A. Cutaneous tissue repair: basic biologic considerations.I J Am Acad Dermatol. 1985; 13: 701-725Abstract Full Text PDF PubMed Scopus (502) Google Scholar). Growth factors and cytokines that are stored in keratinocytes and platelets and are released upon cutaneous injury, like IL-1α (Wood et al., 1996Wood L.C. Elias P.M. Calhoun C. Tsai J.C. Grunfeld C. Feingold K.R. Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis.J Invest Dermatol. 1996; 106: 397-403Crossref PubMed Scopus (230) Google Scholar), are key components in the initiation of wound repair and stimulate formation of a downstream cascade of cytokine release and adhesion molecule expression. As a result, mitotic activity and mobility of dermal and epidermal cells is greatly enhanced (McKay and Leigh, 1991McKay I.A. Leigh I.M. Epidermal cytokines and their roles in cutaneous wound healing.Br J Dermatol. 1991; 124: 513-518Crossref PubMed Scopus (180) Google Scholar;Grinnell, 1992Grinnell F. Wound repair, keratinocyte activation and integrin modulation.J Cell Sci. 1992; 101: 1-5PubMed Google Scholar;Elias et al., 1996Elias P.M. Ansel J.C. Woods L.D. Feingold K.R. Signaling networks in barrier homeostasis. The mystery widens.Arch Dermatol. 1996; 132: 1505-1506Crossref PubMed Google Scholar;Martin, 1997Martin P. Wound healing-aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3410) Google Scholar). During this repair process the wound gap that is temporarily shielded by a clot is replaced by new tissue. In order to regenerate a functional replacement, proliferation, migration, and differentiation of all cell types involved must be tightly controlled. Such a control is at least in part exerted by cytokines and growth factors (Martin, 1997Martin P. Wound healing-aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3410) Google Scholar). Members of the epidermal growth factor (EGF) and fibroblast growth factor (FGF) family are important key regulators of keratinocyte proliferation and migration at the wound edge and their exogenous application exhibits beneficial effects on impaired wound healing (Davidson et al., 1985Davidson J.M. Klagsbrun M. Hill K.E. Buckley A. Sullivan R. Brewer P.S. Woodward S.C. 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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 (511) Google Scholar;Mellin et al., 1995Mellin T.N. Cashen D.E. Ronan J.J. Murphy B.S. DiSalvo J. Thomas K.A. Acidic fibroblast growth factor accelerates dermal wound healing in diabetic mice.J Invest Dermatol. 1995; 104: 850-855Abstract Full Text PDF PubMed Scopus (61) Google Scholar). The beneficial effect of exogeneous transforming growth factor (TGF)-β in treatment of impaired wound healing is also well known (Pierce et al., 1989Pierce G.F. Mustoe T.A. Lingelbach J. Masakowski V.R. Gramates P. Deuel T.F. Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats: possible regulation in macrophages by platelet-derived growth factor.Proc Natl Acad Sci USA. 1989; 86: 2229-2233Crossref PubMed Scopus (259) Google Scholar;Beck et al., 1993Beck L.S. DeGuzman L. Lee W.P. Xu Y. Siegel M.W. Amento E.P. One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing.J Clin Invest. 1993; 92: 2841-2849Crossref PubMed Scopus (210) Google Scholar). Recently, rapid upregulation of TGF-β1, -β2, and -β3 as well as of other members of the TGF-β superfamily, like activin in skin wounds, has been demonstrated, although the function of distinct members of the TGF-β superfamily in the process of wound healing remains to be unraveled (Frank et al., 1996Frank S. Madlener M. Werner S. Transforming growth factors beta1, beta2, and beta3 and their receptors are differentially regulated during normal and impaired wound healing.J Biol Chem. 1996; 271: 10188-10193Crossref PubMed Scopus (310) Google Scholar;Hübner and Werner, 1996Hübner G. Werner S. Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes.Exp Cell Res. 1996; 228: 106-113Crossref PubMed Scopus (69) Google Scholar;Hübner et al., 1996Hübner G. Hu Q. Smola H. Werner S. Strong induction of activin expression after injury suggests an important role of activin in wound repair.Dev Biol. 1996; 173: 490-498Crossref PubMed Scopus (164) Google Scholar). We have recently shown that the tight regulation of expression of bone morphogenetic protein-6 (BMP-6), a member of the TGF-β superfamily, is important for maintaining skin homeostasis (Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). In murine skin BMP-6 is first expressed at day 15.5 p.c. with the onset of stratification in the suprabasal layers of the epidermis. Expression persists at high levels in perinatal epidermis and declines after 6 d p.p. to a very low level in adult skin (Lyons et al., 1989Lyons K.M. Pelton R.W. Hogan B.L. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (392) Google Scholar;Wall et al., 1993Wall N.A. Blessing M. Wright C.V. Hogan B.L. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).J Cell Biol. 1993; 120: 493-502Crossref PubMed Scopus (111) Google Scholar;Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). Thus, high levels of epidermal expression of BMP-6 coincide with a period of most active proliferation in the epidermis, the onset of stratification, and the rapid growth of the body surface at late stages of development and shortly after birth. In the adult skin, epidermal proliferation is maintained at a moderate level and concomitantly, expression levels of BMP-6 are low (Lyons et al., 1989Lyons K.M. Pelton R.W. Hogan B.L. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (392) Google Scholar;Wall et al., 1993Wall N.A. Blessing M. Wright C.V. Hogan B.L. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).J Cell Biol. 1993; 120: 493-502Crossref PubMed Scopus (111) Google Scholar;Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). Dysregulation of expression of BMP-6 in transgenic mice leads to a dramatic change in epidermal cell proliferation and differentiation. Strong and uniform overexpression of BMP-6 in suprabasal epidermis inhibited keratinocyte proliferation in neonates, whereas a weak and patchy pattern of expression of BMP-6 induced epidermal hyperproliferation in postnatal skin (Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). As the arrangement of developing tissues and the control of repair processes both require coordination of proliferation and differentiation by spatial confinement of growth factor activity (Cross and Dexter, 1991Cross M. Dexter T.M. Growth factors in development, transformation, and tumorigenesis.Cell. 1991; 64: 271-280Abstract Full Text PDF PubMed Scopus (598) Google Scholar), we analyzed the expression of BMP-6 in adult wounded skin. We could demonstrate strong induction as well as transcriptional and post-transcriptional control mechanisms regulating the expression of BMP-6 in the epidermis of mice in a temporally and spatially defined manner during wound healing. In human skin ulcerations we also found upregulation of BMP-6 but a loss of the temporal and spatial regulation that in normally healing skin wounds confines the synthesis of BMP-6 to the outermost epidermal layers distal to the wound margins. Therefore, the loss of control mechanisms and the resulting panepidermal expression of BMP-6 may be causally involved in the inhibition of reepitheliazation in skin ulcers. To test this hypothesis we investigated the process of wound healing in transgenic mice that are overexpressing BMP-6 in suprabasal layers of adult skin. Mice of strain FVB/N and mice of the BMP-6 transgenic line VI-6–32, which were also maintained on a FVB/N genetic background, as well as transgenic and normal F1 crosses to C57Bl6 were used (Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). For wound healing studies transgenic males and nontransgenic control males from the same colony aged 6–8 mo were caged individually. The animals were anethesized by i.p. injection of avertin and shaved on their middorsum. Full-thickness incisional or excisional wounds of 8 mm length or diameter, respectively, were set middorsally with a pair of scissors. After surgery mice were caged individually. In one set of experiments the degree of wound healing in control and transgenic mice was monitored by visual inspection and photography in 1 or 2 d intervals. In another set of experiments mice were sacrificed and wound areas were removed at days 1, 3, 5, 7, 13, 25, and 42 after injury. For cell proliferation assays bromodeoxyuridine (BrdU) [0.25 mg per g body weight in phosphate-buffered saline (PBS)] was injected i.p. 2 h before the animals were sacrificed. Each wound was disected in the midtransversal plane and one half was fixed in methanol/dimethylsulfoxide (4:1) and embedded in paraffin. From embedded tissues, 4 μm sections were cut for hematoxylin and eosin staining. The other half was embedded in tissue freezing medium (Jung; Leica Instruments, Nuβloch, Germany) and frozen in liquid nitrogen for immunohistology andin situ hybridization experiments. Five micrometer sections were cut perpendicular to the longitudinal direction incisional wounds. In order to study BMP-6 expression in human wounds, six chronic leg ulcers were examined, including diabetic ulcers, venous ulcers, and post-thrombotic ulcers as well as ulcers resulting from arterial occlusion. Specimens were taken from the edge of the ulcer comprising the bed of the wound and the surrounding epithelium. Samples from normally healing wounds were biopsied from Syspurderm covered large skin wounds at the uncovered wound margins. Tissue samples were frozen in liquid nitrogen. For BMP-6 immunohistochemistry we used an affinity purified polyclonal antibody (Wall et al., 1993Wall N.A. Blessing M. Wright C.V. Hogan B.L. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).J Cell Biol. 1993; 120: 493-502Crossref PubMed Scopus (111) Google Scholar) at a 1:200 dilution. Secondary antibodies were F(ab)2 fragments conjugated with alkaline phosphatase or peroxidase in a 1:2000 and a 1:50 dilution, respectively (Boehringer, Mannheim, Germany). Tissue sections were fixed in aceton at 4°C for 10 min. For alkaline phosphatase staining cryostat sections were preblocked in 1% bovine serum albumin, 5% fetal calf serum, 0.5% Tween 20, 0.1 M MgCl2 in 10 mM Tris-HCl (pH 7.4). Endogeneous phosphatase activity was inhibited by addition of 1 mM levamisol in the substrate solution. For peroxidase staining sections were incubated in 0.6% hydrogen peroxide in methanol to quench endogeneous peroxidase activity and preblocked in 5% bovine serum albumin/1% goat serum in PBS. For staining of macrophages we used a rat monoclonal antibody against murine CD11b (M1/70; PharMingen, Hamburg, Germany) at a 1:50 dilution in blocking buffer (5% goat serum, 1% bovine serum albumin). Rhodamine-labeled goat anti-rat secondary antibody (Dianova, Hamburg, Germany) was applied in a 1:800 dilution after 2 h preabsorption to mouse liver powder. Incorporation of BrdU into DNA of proliferating cells was visualized by staining of cryostat sections with fluorescein-conjugated anti-BrdU monoclonal antibodies (Boehringer), following the supplier's protocol with the modification that cryostat sections were fixed for 15 min in freshly prepared 4% paraformaldehyde in PBS. BMP-6 specific cDNA probes were generated by polymerase chain reaction. The primer pair for BMP-6 consisted of primer hB6–3 (agggatccCAACAGAGTCGTAATCGC; capital letters denote nucleotides coding for amino acid 382–387 in the human cDNA;Celeste et al., 1990Celeste A.J. Iannazzi J.A. Taylor R.C. Hewick R.M. Rosen V. Wang E.A. Wozney J.M. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone.J Biol Chem. 1990; 265: 21720-21726PubMed Google Scholar) and of primer hB6–5 (agataagcttGTTGCATTCATGTGTGCG; capital letters denote nucleotides coding for amino acid 455–451 in the human cDNA;Celeste et al., 1990Celeste A.J. Iannazzi J.A. Taylor R.C. Hewick R.M. Rosen V. Wang E.A. Wozney J.M. Identification of transforming growth factor beta family members present in bone-inductive protein purified from bovine bone.J Biol Chem. 1990; 265: 21720-21726PubMed Google Scholar). Fragments were cloned into pGEM-4Z (Promega, Heidelberg, Germany) and digoxigenin-labeled sense and anti-sense RNA was generated usingin vitro transcription with SP6 and T7 RNA polymerases according to the supplier's protocol (Boehringer). Cryosections were cut at 5 μm thickness and transferred onto polylysine coated DEPC-treated slides and fixed immediately after sectioning in freshly prepared 4% paraformaldehyde in PBS for 20 min. Sections were acetylated in 0.1 M triethanolamine/0.2% acetic anhydride for 10 min, washed in PBS, dehydrated in graded alcohol, and air dried. Prehybridization and hybridization were performed at 38°C. The hybridization mixture contained 100 pg digoxigenin-labeled RNA per ml, 50% deionized formamide, 8% dextrane sulfate, 4×sodium citrate/chloride buffer, 1×Denhardt's mixture, 0.5 mg salmon sperm DNA per ml. The slides were washed several times in 2×sodium citrate/chloride buffer, digested with RNAse A (20 μg per ml) for 30 min at 37°C, washed three times in 60% deionized formamide/2×sodium citrate/chloride buffer at 38°C and several times in Tris buffer (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) at room temperature. Sections were blocked for 30 min in 3% blocking mixture (Boehringer) before incubation with anti-DIG alkaline phosphatase-conjugated F(ab)2 fragments (Boehringer; 1:200 dilution in 3% blocking mixture) at room temperature for 2 h. Signal detection was according to protocols from Boehringer by using NBT/BCIP as substrate. Endogeneous alkaline phosphatase was inhibited by addition of 1 mM levamisol to the substrate solution. In order to study expression of BMP-6 in wound tissue on the protein level, we collected samples of full-thickness incisional wounds of mouse skin at day 1, 3, 5, 7, 13, 25, and 42 post-wounding and performed immunostainings with a BMP-6 specific polyclonal anti-serum. Up to 2 d after wounding BMP-6 was hardly detectable at the wound margin (Figure 1a) or in the wound periphery as it has been described for normal skin of adult mice (Lyons et al., 1989Lyons K.M. Pelton R.W. Hogan B.L. Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (392) Google Scholar;Wall et al., 1993Wall N.A. Blessing M. Wright C.V. Hogan B.L. Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).J Cell Biol. 1993; 120: 493-502Crossref PubMed Scopus (111) Google Scholar;Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). Three days after wounding a dramatic upregulation of BMP-6 was observed in regenerating epidermis proximal to the wound edge (Figure 1b). Outer root sheath cells of hair follicles adjacent to the wound edge also stained positive for BMP-6 (Figure 1b). Distal to the wound towards the normal nonregenerating epidermis immunoreactivity against BMP-6 gradually diminished. BMP-6 induction was not confined to epidermal cells of the wound area. Significant immunostaining was also detected in fibroblast-shaped cells present in the wound space (Figure 1c). Strong reactivity for BMP-6 was seen 5–7 d after incision in the wound periphery and in keratinocytes of the newly formed epithelium that covered the wound (Figure 1d). High protein levels were still detected 13 d after wounding at the wound margins and in the newly formed epithelium, including the merging wedges of keratinocytes (Figure 1e,f). Even 25 d after wounding elevated reactivity against BMP-6 was observed in the newly formed epidermis covering the incision, whereas after 42 d BMP-6 protein was hardly detectable any more (data not shown). In all cases, synthesis of BMP-6 was confined to suprabasal keratinocytes with strongest accumulation in the upper granular layer (Figure 1a–f). In situ hybridization experiments performed with a BMP-6-specific cRNA probe and immunohistochemistry demonstrated striking post-transcriptional regulation of BMP-6 expression in mouse skin after incisional wounding. BMP-6-specific RNA was rapidly induced after wounding in all keratinocytes of the thickened epidermis adjacent to the wound site, including the basal epidermal layer within 24 h (Figure 2a). This distribution pattern was in contrast to normal skin from neonatal mice where accumulation of BMP-6-specific RNA was confined to suprabasal epidermal layers (insert inFigure 2a). Uniform distribution of BMP-6-specific RNA was maintained throughout all layers of newly formed wound epithelium including the leading edge of migrating keratinocytes until the end of reepitheliazation around 13 d after incision (Figure 2b,c). When epidermal thickness in the wound area decreased, BMP-6-specific RNA was still present but became restricted to the suprabasal layers (Figure 2d). In contrast to the rapid induction of BMP-6-specific RNA, protein was not detected at all in hyperplastic epidermis flanking the 1 d old wound (Figure 2e). Significant upregulation of BMP-6 protein beginning with day 2–3 after incision (Figure 1b) remained restricted to the uppermost suprabasal layers at the wound periphery and in the newly formed epithelium (Figure 2f–h). BMP-6 protein was not expressed at the tip of the migrating edge of keratinocytes (Figure 2f). In normal human skin BMP-6 RNA and protein are expressed by suprabasal layers of epidermis at a very low level that is hardly detectable byin situ hybridization and immunohistochemistry. We studied expression of BMP-6 on the RNA or protein levels in the epithelium of human skin wounds. Sections from chronic skin ulcers from six patients with different etiology were analyzed byin situ hybridization and immunohistochemistry. In all lesions examined a prominent staining for BMP-6-specific RNA was detected throughout the epidermis at the wound margin (Figure 3a). Even basal keratinocytes at the wound edge expressed BMP-6 on the RNA level (Figure 3b). On the protein level, BMP-6 was strongly expressed, frequently encompassing all the suprabasal layers of the epidermis at the wound margin (Figure 3c). Occasionally the pattern of protein staining was patchy (data not shown). In contrast to BMP-6-specific RNA, protein was not detected in basal keratinocytes at the leading edge of the migrating epithelium, which were in direct contact to the extracellular matrix (Figure 3d). In normally healing human skin wounds, BMP-6 is not found on the protein level in keratinocytes migrating into the wound bed but is restricted to the wound margins in a manner comparable with normally healing murine wounds (Figure 3e). As BMP-6-specific RNA and protein could be shown to be strongly upregulated in nonhealing wounds of different patients, we examined whether constitutive overexpression of BMP-6 in mouse skin will impede wound healing. Repair of incisional and excisional wounds was compared between normal mice and transgenic mice overexpressing BMP-6 in the suprabasal layers of the epidermis by visual inspection (Figure 4). Full-thickness excisional wounds of around 8 mm diameter on the back skin of four BMP-6 transgenic mice and four control mice were photographed at different time points after injury. Up to 2 d after incision wounds of transgenics and control mice looked similar (Figure 4). Wounds were covered with a dehydrated wound crust the day after surgery. Transgenic wounds sometimes showed deposits of dried blood at the wound margins resulting from enhanced angiogenesis in transgenic skin (Blessing et al., 1996Blessing M. Schirmacher P. Kaiser S. Overexpression of bone morphogenetic protein-6 (BMP-6) in the epidermis of transgenic mice: inhibition or stimulation of proliferation depending on the pattern of transgene expression and formation of psoriatic lesions.J Cell Biol. 1996; 135: 227-239Crossref PubMed Scopus (119) Google Scholar). Wound crusts of control mice showed first signs of detaching 6 d after incision. Between days 9 and 13 after surgery all control mice had lost the scab, sometimes revealing a small residual defect, whereas transgenic wounds retained their scab till day 16–20. Three weeks after incision all wounds were fully closed (Figure 4). A similar delay of wound repair was observed for incision wounds in BMP-6 transgenics (data not shown). To analyze the delay in the repair process of transgenic skin wounds in more detail, full-thickness incisional wounds in the back skin of BMP-6 transgenic and control mice were examined histologically. Wound tissue was removed from four mice of each group at days 1, 3, 5, 7, and 13 after injury. By histology, no significant differences in the initial wound healing response of clotting and granulation tissue formation were seen between transgenic mice and controls (data not shown). But examination of the 7 d and 13 d old wounds revealed a consistent delay of the repair process in wounds of BMP-6 transgenics (Figure 5). Seven days after wounding migrating keratinocytes had covered the entire wound in most of the control mice. The wound was filled by a thick layer of granulation tissue and the wound crust was already detaching (Figure 5a,a′). After 13 d a fully stratified epidermis was present in all controls and the granulation tissue was diminished. Formation of fibrous bundles at the wound margins indicated incipient r