Präsident der Humboldt-Universität zu Berlin

Prof. Dr. Christoph Marschkies

• Preface

• Zusammenfassung

• Introduction

  • Wound healing in the skin

  • Mammalian skin

  • The tyrosine kinase receptor Met

  • Met signal transduction

  • Met signalling during development

  • Met function in the adult

  • The aim of the study

• Results

  • Expression of Met and HGF/SF in the skin and during skin wound healing

  • Generation of mice deficient in Met in keratinocytes

  • Met signaling during generation and maintenance of the skin

  • Wound closure in conditional Met mutant mice

  • Contribution of cells in the hyperproliferative epithelium

  • Scratch-wound healing of Met mutant keratinocytes in cell culture

  • Cytoskeleton rearrangement in cultured scratchwounded keratinocytes

  • Signal transduction in primary keratinocytes

• Discussion

  • Conditional mutagenesis to investigate Met function in the skin

  • The role of the tyrosine kinase receptor Met in the skin

  • Only non-recombined cells contribute to wound healing

  • The role of HGF/SF and Met in development and regeneration

  • Only Met-positive keratinocytes contribute to healing of scratch-wounds in vitro

  • The Met receptor as a therapeutically target

• Materials and Methods

  • Extraction and purification of DNA

  • Polymerase chain reaction (PCR)

  • Southern blotting

  • Cell culture

  • Wounding of skin

  • Immunhistochemical techniques

  • Protein biochemistry

• Abbreviations

• References

• Erklärung

• Publications

• Figure 1. Scheme of different stages of wound repair in mammals A: 12–24 h after injury the wounded area is filled with a blood clot. B: at days 3–7 after injury, endothelial cells migrate into the clot; they proliferate and form new blood vessels. Fibroblasts migrate into the wound tissue, where they proliferate and form extracellular matrix. The new tissue is called granulation tissue. Keratinocytes proliferate at the wound edge and migrate down the injured dermis and above the provisional matrix. C: 1–2 wk after injury the wound is completely filled with granulation tissue. Fibroblasts have transformed into myofibroblasts, leading to wound contraction and collagen deposition. The wound is completely covered with a neoepidermis. Modified from Werner and Grose, 2003.

• Figure 2. Chronic wound. Keratinocytes at the edge of the wound (purple) are hyperproliferative (indicated by mitotically active cells present throughout the suprabasal layers), hyperkeratotic (indicated by thick cornified layer) and parakeratotic (indicated by presence of nuclei in the cornified layer). BM=basement membrane.

• Figure 3. Mammalian skin and its appendages. Skin consists of the epidermis and dermis, separated by a basement membrane (BL). The epidermis is composed of is the basal layer (BL), differentiated spinous layer (SL), granular layer (GL) and the stratum corneum (SC). Also shown is a cross-section of a hair follicle, which consists of an outer root sheath that is contiguous with the basal epidermal layer, the hair bulb, made from proliferating matrix cells, and the bulge, which is part of the outer root sheath and is where epidermal stem cells reside. Modified from Fuchs and Raghavan, 2002.

• Figure 4. Docking sites of Met. Shown are the phosphotyrosines binding sites of Met, as well as their direct interaction partners. α and β refer to the subunits of the receptor. Gab1, growth-factor-receptor-bound protein 2 (Grb2)-associated binder 1; HGF/SF, hepatocyte growth factor/scatter factor; PI3K, phosphatidylinositol 3-kinase; PLCγ, phospholipase Cγ; Shc, Src-homology-2 (SH2)-domain-containing; Shp2, SH2-domain containing protein tyrosine phosphatase 2; Grb2 and Grb10, growth-factor-receptor bound-protein 2 and 10; Ship, SH2-domain-containing inositol-5-phosphatase; p85 refers to the regulatory subunit of PI3K. Modified from Birchmeier et al., 2003.

• Figure 5. Signaling by the receptor tyrosine kinase, Met. Upon binding of HGF/SF, Met recruits various adapter proteins like Gab1 and Grb2 and activate Shp2, Ras, Erk and PI3K pathway. These pathways regulate cell adhesion, cytoskeleton, motility, cell cycle and apoptosis (Birchmeier et al., 2003).

• Figure 6. Gab family proteins. Schematic domain structures of mammalian Gab1 proteins, zebrafish Gab1 (zfGab1) and their invertebrate orthologs in Drosophila (DOS) are shown. All Gab family proteins consist of N-terminal pleckstrin-homology (PH) domain, prolinerich domains (P) and multiple tyrosines. The unique Met-binding site (MBS) that allows for direct interaction with Met is also indicated.

• Figure 7. Migrating muscle precursor cells in HGF/SF/ and Met/ embryo. During normal development muscle precursor cells (red) delaminate from the epithelial dermomyotome (Dm, blue) and migrate to the limb bud, where they differentiate into myoblasts. In HGF/SF–/– or Met–/– embryos, the progenitor cells are not released, but remain in the dermomyotome. HGF/SF, hepatocyte growth factor/scatter factor; My, myotome; Sc, sclerotome. Modified from Birchmeier and Brohmann, 2000.

• Figure 8. Expression of the Met tyrosine kinase receptor and its ligand, HGF/SF, in the skin. A. Immunofluorescence staining using Met antibody on a tail skin section. Met is detectable in the epidermis. B. Immunofluorescence staining using HGF/SF antibody on a tail skin section, showing HGF/SF protein expression in the dermis, associated with loosely packed fibroblasts. C. Hematoxylin/eosin staining on a tail skin section. This staining helps to recognise the morphology of tail skin. The round structure in the dermis is a hair follicle.

• Figure 9. Expression of activated Met in the skin. A, B Immunostaining of skin section of the age P32 (A) and P5 (B) with anti phosphoMet (red) and anti CD34 (green) antibodies. CD34 is a marker of hair bulge stem and hematopoietic cells. The merged fluorescence shows that Met and CD34 are colocalized in the bulge stem cells. Scale bar 20μm

• Figure 10. Generation of skinspecific Met mutant mice. A. Schematic representation of nonrecombined and recombined alleles of Met. Exon 15 of the Met gene that encodes the ATP-binding site (red box) was flanked by loxP sites (triangles) and is excised after K14cre-induced recombination. Blue boxes indicate exons 14 and 16. The sizes of the restriction fragments generated by BamHI digest before and after recombination are indicated. B, BamHI; P, Pst. B. Southern blot analysis of epidermis from control and conditional Met mutant mice of different ages (12 weeks old, E17.5, P8). C. Southern blot analysis of different organs of conditional Met mutant mice.

• Figure 11. Expression of K14cre in the skin using Z/AP reporter mice. A. Double staining of skin section from Z/AP; K14cre mice for lacZ (blue, nonrecombined) and alkaline phosphatase activity (yellow, recombined). B. A higher magnification shows an area of nonrecombined epidermal keratinocytes (blue patch, marked by arrow). CD. Higher magnifications show also two independent hair follicles. Arrector pilli muscle cells, which surrounded the follicle, are nonrecombined (blue). Scale bar, 50μm (A, B), 20μm (C, D).

• Figure 12. Hair follicle cycle in control and conditional Met mutant mice. Sagittal sections of control and conditional Met mutant skin stained with hematoxylin/eosin at P1 (first anagen), P5 (first anagen), P8 (first anagen), P18 (first catagen), P20 (first telogen), P30 (second anagen).

• Figure 13. Immunohistological analysis of the skin in conditional Met mice A. Immunohistological staining for keratin 10 and keratin 6 on dorsal skin paraffin sections of 2 months old mutant and control mice. Keratin 6 is constitutively expressed in the outer root sheath of hair follicles and is observed in both, control and mutant mice. Keratin 10 is present in the differentiated, upper layers of the epidermis. B. Immunohistological staining for loricrin on dorsal skin sections of control and mutant mice. Loricrin is expressed in upper layers of the epidermis. There are no differences in expression of these proteins in the skin between mutant and control.

• Figure 14. Expression of HGF/SF and Met during wound healing. A. Scheme of an entire wound 3 days after injury. Keratinocytes (red) at the wound edge proliferate and migrate down the injured dermis to form the socalled hyperproliferative epithelium (HE, marked by arrow). G, granulation tissue; D, dermis; F, fatty tissue; Es, eschar. B and C. In situ hybridisation of wounded skin with HGF/SF probe 1 day (B) and 3 days (C) after injury. HGF/SF is expressed in the dermis close to the clot at day 1. At day 3 after wounding, HGF/SF is highly expressed in the hyperproliferative epithelium (HE). E and F. In situ hybridization with the Met probe 1 day (E) and 3 days (F) after injury. Met is expressed in the epidermis and in the hyperproliferative epithelium (HE) at day 3 following wounding. D and G In situ hybridization with sense probes of HGF/SF (D) and Met (G). Scale bar, 50μm

• Figure 15. Expression of K14cre during wound healing. A. Double staining of alkaline phosphatase and βgalactosidase activity of wound section from Z/AP; K14cre mice. K14cre-induced recombination is observed in the unwounded epidermis and in the hyperproliferative epithelium. B. Immunohistological analysis of a wound section from control mice using antibodies directed against keratin 14 (red) and fibronectin (green). Scale bar, 100μm

• Figure 16. Wound healing in conditional Met mutant mice. A and D. Hematoxylin/eosin staining of sections of wound from control and mutant mice 3 days (A) and 5 days (C) after wounding. B and E Masson trichrome staining of sections 3 days (B) and 5 days (D) after injury. C and F. Immunofluorescence staining for Keratin 6 (red) and fibronectin (green) from control and mutant mice 3 days (E) and 5 days (F) after injury. Arrows indicate the hyperproliferative epithelium (HE). F, fatty tissue; G, granulation tissue; Es, eschar; HF, hair follicle Scale bar, 100μm

• Figure 17. Quantification of wound healing in control and conditional Met mutant mice A. Wound closure kinetics in control and mutant mice. B. Quantification of the area of hyperproliferative epithelium 3, 5 and 7 days after wounding in control and mutant mice; only sections of the middle of the wounds were used for quantification. C. Proliferation of keratinocytes in the hyperproliferative epithelium from control and mutant mice 3, 5, and 7 days after wounding, as assessed by the proportion phosphohistone 3positive nuclei in the epithelium. Error bars represent standard deviations. A Student’s test was performed, and significant differences between control and mutant was observed 3 days after injury, P value, p=0.01. D. Proliferation of keratinocytes in the hyperproliferative epithelium from control and mutant mice 3, 5, and 7 days after wounding, as assessed by the proportion of BrdUpositive nuclei in the epithelium. Significant statistical differences between control and mutant was observed 5 days after injury, P value, p=0.01. E. Number of cells in the hyperproliferative epithelium quantified as Yopropositive cells for control and mutant 3, 5 and 7 days after injury. Yopro is a nuclear dye. F. Quantification of BrdUpositive cells in the hyperproliferative epithelium of control and mutant mice at different time points after injury.

• Figure 18. Only residual Met positive keratinocytes contribute to the hyperproliferative epithelium of wounds in conditional Met mutant mice A, B. Isolation of hyperproliferative epithelium by laser capture microdissection. A wound section before (A) and after laser capture microdissection (B) is shown. C. Southern blot analyses of back epidermis and hyperproliferative epithelia from conditional Met mutant mice. Microdissected hyperproliferative epithelia of wounds 3 days (middle) and 5 days (right) after injury were collected. Southern blotting of two preparations from different pools of microdissected tissues is shown. The hyperproliferative epithelium 5 days after injury in conditional Met mutant mice is formed exclusively by cells, which contain the nonrecombined Metflox allele. At day 3, a 1:1 mixture of recombined and nonrecombined cells are seen (middle).

• Figure 19. Only phosphoMet positive cells contribute to the hyperproliferative epithelium in control and mutant. Immunohistological analysis of wound sections from control and conditional mutant mice using anti-phosphoMet antibodies (red immunofluorescence in A and B, and brown immunohistochemistry in C and D). Cells in the hyperproliferative epithelium of conditional Met mutant mice (outlined) are phosphoMet positive. Arrows mark phosphoMet positive cells in the lower hyperproliferative epithelia layers. Scale bar, 100μm

• Figure 20. Isolated keratinocytes from control and conditional Met mutant mice. A. Immunostaining with anti keratin 14 antibodies on primary keratinocytes isolated from the skin of control and conditional Met mutant mice. Scale bar, 20μm B. Nothern blot analysis of control keratinocytes stimulated with HGF/SF probed with Met.

• Figure 21 Scratchwound healing in cell culture of primary keratinocytes: Primary keratinocytes were isolated from newborn skin of control (A) and conditional Met mutant mice (BC). After scratchwounding, cells were further cultured in the presence of HGF/SF or TGFα. Photos were taken 0, 24, 48 and 96 hours after scratchwounding. Wounds in the cultures derived from conditional mutant mice did only close after 96 hours in the presence of HGF/SF. Scale bar, 100μm. D. Proliferation of primary keratinocytes from control and conditional Met mutant mice 24 hours after stimulation with HGF/SF, as assessed by phosphohistone 3 antibody staining (red). A dashed line marks the scratch edge. Counterstaining was performed with phalloidin (green). Scale bar, 100μm E. Quantification of proliferation of primary keratinocytes at wound edges stimulated with HGF/SF in the experiments described in D. Error bars represent standard deviations.

• Figure 22 Only Met positive primary keratinocytes migrate into the scratchwounds in cell culture. A, B. Primary keratinocytes isolated from control (A) and conditional Met mutant (B) skin were scratchwounded and further cultured with HGF/SF. After 24, 48 and 96 hours cells were stained with phosphoMet antibodies (green). Nuclei were visualised by Yopro staining (red). In mutant cell population, phosphoMet containing cells were initially rare, but finally, after 96 hours, occupied the entire scratched area. The original edges of the scratchwounds are marked with a dashed line. Scale bar, 50μm

• Figure 23. Met mutant keratinocytes are unable to rearrange their focal contacts and their cytoskeleton at the scratchwound edges following HGF/SF treatment Keratinocytes derived from control and conditional Met mutant mice were stained 24 hours after scratchwounding with antibodies directed against vinculin A., with phalloidin A, D, antibodies directed against VASP B, paxillin C, RhoA D and γtubulin E. Arrows mark the newly formed focal contacts (AC) and RhoA at the rear of the cells (D). Arrowheads mark cytoplasmatical and perinuclear localization of RhoA in mutant. The dotted line indicates the edges of the wounds. Scale bar, 50μm (AD), 20μm (E).

• Figure 24 Signaling is blocked in keratinocytes derived from conditional Met mutant mice that are treated with HGF/SF, but not with TGFα. A. Western blot analysis of phospho Erk1/2, total Erk1/2, phospho Akt, total Akt, phospho Gab1 and phospho PAK1/2 in keratinocytes derived from control and conditional Met mutant mice. Cells were stimulated with HGF/SF or TGFαfor 0, 10 or 30 minutes. Erk1/2, Akt, Gab1 and PAK1/2 are not activated (phosphorylated) in cultured keratinocytes from the conditional mutant mice after HGF/SF stimulation. B. Quantification of the phosphoPAK1/2 signal on Western blots (A) as assessed by pixel intensity.