Collagen-receptor signaling in health and disease

ARTICLE

The ability to synthesize extracellular matrix (ECM) was an essential stepping stone during the early evolution of multicellular life. Synthesis and degradation of ECM proteins are critical not only for proper embryonic and postnatal development but throughout adulthood. In turn, any aberrant communication between cells and their surrounding ECM can lead to a plethora of human diseases, for example, atherosclerosis, arthritis, renal or lung fibrosis, and cancer. This review is an up to date perspective of the multiple proteins and pathways involved in cell-ECM signaling, focusing on collagens and their receptors. The term "signaling" shall include all forms of cell/cell and cell/ECM communication, leading to a wide array of different cellular responses, including adhesion, migration, invasion, proliferation, growth arrest or apoptosis.

Cell-matrix contacts: a fresh look

In the past years, much has been learned about the structure and function of ECM receptors. A growing number of distinct matrix receptor classes have been described [1]. Evidence is accumulating that complexes between receptors and their ECM ligands do not appear to be a one to one reaction, instead clusters of multiple ligands and signaling receptors interact with each other. Specific cellular responses result from the integration of multiple incoming signals. The best characterized matrix receptors are the integrins, but syndecans, receptor tyrosine kinases (RTK) and phosphotyrosine phosphatases as well as heparan sulfate receptors and hyaluronan receptors have recently been shown to be equally important players. Additionally, the number of genes coding for collagens has been steadily increasing. Currently, at least 24 different types of collagens are known, giving rise to at least 50 homo- or heteromeric triple-helical molecules.

Collagen-signaling by integrins

Integrins are heterodimeric proteins consisting of two non-covalently bound subunits, named alpha and beta subunit. In the human genome, 24 alpha chains and 9 beta chains have been found [2]. From approximately 200 heterodimers that could form theoretically, only about 25 have been identified so far in vivo. Integrins are receptors essential for cellular adhesion to a huge variety of ECM proteins, including vitronectin, fibrinogen, fibronectin, osteopontin, collagens, laminins, tenascins and thrombospondins or to cellular receptors, such as VCAM-1 and ICAM [3]. Only 4 of them have been shown to bind collagens. These integrin collagen-receptors have the beta1 subunit in common which can pair up with one of the following alpha subunits: alpha1, alpha2, alpha10 and alpha11. The 4 integrins are not only receptors for collagens but at least alpha1, alpha2 also bind laminins, albeit with much lower affinity. Whereas the alpha1beta1 receptor preferably binds to basement membrane (type IV) and type XIII collagens, alpha2beta1 and alpha11beta1 have a preference for fibrillar collagens and alpha10beta1 integrin for type II collagen [4-11].

A common theme amongst the extracellular domain of the alpha1, -2, -10 and -11 subunit is an app. 200 amino acid long homology region, called I domain, which is responsible for collagen-binding. The I domain is related to the von Willebrand factor domain which is commonly found in cell adhesion receptors and ECM proteins. Electron microscopy of the extracellular part of the alpha integrin suggested an N-terminally globular I domain which is followed by an extended stalk region [12-14]. Molecular proof of the collagen-integrin complex has been obtained from the co-crystallization of the I domain of alpha2 integrin with a triple-helical collagen-related peptide [15]. The structure reveals the molecular binding epitopes of the collagen-integrin interaction. A binding epitope, formed by three alpha helices and a central beta-sheet on the surface of the I domain, recognizes the hexapeptide motive Gly-Phe-Hyp-Gly-Glu-Arg of the collagen-like peptide. The glutamate in this peptide binds a divalent cation, normally magnesium, which is complexed by the I domain. Collagen-induced complexation of the metal cation does not only induce a conformational change in the I domain but in the entire heterodimeric receptor, which allows signal transduction from the extracellular space into the cytoplasm. However, it remains unclear if natural collagen molecules are complexed by the I domains in a comparable fashion and whether the alpha subunits of other integrins show similar binding preferences. In vitro, high-affinity binding to collagen was found with purified extracellular regions of the alpha1 and alpha2 subunit, but not with alpha3, suggesting that alpha3beta1, that was originally found to interact with collagen but lacks the I domain, is not a collagen-binding integrin. Furthermore, very recent data suggest that alphaXbeta2 integrin expressed on monocytes can function as type I collagen-receptor as well [16].

Alpha1beta1 is the primary basement membrane collagen-binding integrin, and is therefore expressed in many cell types including smooth muscle cells, endothelial cells and neural crest-derived cells [17, 18]. Alpha2beta1 also has a wide expression in cells and tissues and appears as a central regulator of cell migration, invasion and tissue repair [19]. During mouse development, alpha2 mRNA is particularly expressed during blastocyst implantation and bone remodeling [20]. Moreover, blocking experiments with antibodies against alpha2beta1 or data from patients with a reduced expression of this integrin show a role as collagen-binding receptor in platelet adhesion [21]. Surprisingly however, mice with a platelet-specific ablation of beta1 integrin have essentially normal platelet adhesion and thrombus formation [22]. This integrin is also responsible for controlling protease production and secretion and for mediating extravascular trafficking of neutrophils during inflammatory responses [23, 24]. Together, alpha1beta1 and alpha2beta1 seem to be key regulators in the contraction of collagen networks by fibroblasts, melanoma cells and a number of other cell types [25-27]. Activated hepatic stellar cells, which express very little alpha2beta1 receptor, utilize alpha1beta1 for cell contraction in an in vivo model for liver injury [28]. Collagen-adhesion of vascular smooth muscle cells (VSMC) is mediated by alpha1 and -2 integrins and the proportional expression of individual integrins changes when cells are cultivated on heat denatured versus native collagen [29]. In contrast, alpha10 and -11 seem to be more restrictedly expressed. Whereas alpha10 colocalizes with its ligand, collagen type II, particularly in the cartilage, heart and muscle, alpha11 is found on mesenchymal cells during embryonic development and in muscle tissue in adulthood [8, 30-32].

Do the 4 integrins have overlapping functions or are they functionally distinct receptors for collagen? Data from in vitro and in vivo studies suggest that they have rather distinct specificity, as tissue culture and knockout experiments clearly demonstrate individual roles for each of them (Fig. 1). Whereas all collagen-binding integrins play a role in cell adhesion, their individual functions in collagen degradation and neo-synthesis are rather diverse. Currently, knockout mouse models are available for alpha1, -2, and -10 not yet for alpha11 [33, 34]. Mice lacking integrin alpha1 are viable and show no overt phenotype except that collagen renewal is enhanced in the dermis [35]. However, the in vitro proliferation rate of alpha1-null embryonic fibroblasts is markedly reduced, while cell attachment and spreading are normal [36]. Crossing the alpha1-deficient mice into a mouse strain with collagenase-resistant collagen results in skin thickening [37]. Furthermore, tumors implanted in alpha1 knockout mice are less vascularized, possibly due to an increase in matrix metalloproteinase (MMP) -7 and MMP9 expression which leads to enhanced release of angiostatin [38].

Surprisingly, alpha2 knockout mice show a rather mild phenotype as well (M. Zutter, personal communication). While viability, fertility and wound healing of alpha2 deficient mice is normal, the mammary gland epithelium of adult knockout mice shows a lower degree of branching complexity compared to the wild type. This defect is in line with earlier work showing the importance of this integrin during mammary gland morphogenesis [39]. Because alpha2beta1 is the only collagen-binding integrin in platelets, cell adhesion of platelets from alpha2-null mice is dramatically reduced in vitro which however does not affect platelet coagulation in vivo. Alpha10-deficient mice show no apparent phenotype [34].

Ablation of the beta1 integrin gene in the germline of the mouse gives rise to a more severe phenotype: homozygous embryos arrest at day 4.5 of development. Beta1-null blastocysts show normal development and implant into the uterus, but fail to grow afterwards [40, 41]. The severity of the knockout phenotype might be explained by the fact that beta1 integrin complexes are not only formed with the 4 collagen-binding alpha subunits but with at least 8 other alpha chains which mediate adhesion to and signaling by multiple ECM molecules, other than collagens. Dominant-negative inhibition of beta1 signaling in the mammary gland led to reduced proliferation and impaired differentiation of epithelial cells and allowed the dissection of the signaling pathways downstream of beta1 integrin [42, 43]. To gain further insight into the role of beta1 later in development and adulthood, tissue-specific knockouts have been performed using cre-mediated recombination. Keratinocyte-restricted deletion of beta1 integrin resulted in viable animals showing hair loss, increased dermal fibrosis and blistering at the dermal-epidermal junction due to an instability of the hemidesmosomes [44, 45]. Furthermore, a platelet-specific knockout of beta1 revealed that glycoprotein VI is mainly used for platelet adhesion to collagen, while beta1 has only an adjuvant function [22].

Discoidin domain receptors: the alternative way of collagen-signaling

Like the integrin subunits, discoidin domain receptors (DDR) are molecules with a single transmembrane region. However, in contrast to integrins, DDR belong to the tyrosine kinase receptor family and form homodimers upon ligand engagement (Fig. 1). Sequence prediction methods allow to identify over 90 tyrosine kinases in the human genome, 58 of them being receptor molecules [46]. Two important members of this family, the epidermal growth factor (EGF) receptor and the nerve growth factor receptor have been reviewed earlier in this journal [47, 48]. In the human genome, two genes have been identified that code for discoidin domain receptor-1 and -2 (DDR1 and DDR2). DDR are distinguished from other receptor tyrosine kinases by a discoidin domain in their extracellular domain, which is a homology region originally identified in the protein discoidin I from Dictyostelium discoideum [49-51]. During cell aggregation of Dictyostelium, discoidin I is secreted and functions as a galactose-binding lectin. In slime mold, it is thought to be important in the maintenance of morphology, cytoskeletal organization, and the ability of the cells to align during aggregation.

Various types of collagen are ligands for DDR [52, 53]. Whereas in DDR1, autophosphorylation is achieved with all collagens (type I to type VI and type VIII have been tested so far), DDR2 is only activated by fibrillar collagens, in particular by collagen type I and type III. In contrast to most other tyrosine kinase receptors, maximal activation of DDR requires up to 18 hours stimulation by collagen. The presence of the discoidin domain in DDR1 has been shown to be essential for collagen binding. Yet, the minimal binding sequence in a collagen molecule essential for DDR interaction has not been defined. Furthermore, ligands unrelated to collagen might also bind DDR and activate together with or separately from collagen.

The DDR1 cDNA has been isolated from several human tissues and carcinoma cell lines, notably from MCF7 mammary carcinoma cells, ovarian and esophageal cancer cells, primary pediatric brain tumor samples, and from human keratinocytes and bronchial epithelial cells [51, 54-59]. The various other names originally given to DDR1 and DDR2 have been reviewed elsewhere [60]. Due to its expression in the hematopoetic system, particular in monocyte-derived dendritic cells, DDR1 has been annotated as CD167 [61, 62]. RNA in situ hybridization analysis has shown specific expression of human DDR1 in epithelial cells, particularly in the brain, kidney, lung, mucosa of the colon, the follicles of the thyroid, and the islets of Langerhans [51]. The expression of DDR1 and its ligands in the cerebellum have been analysed in more detail [63]. In the cerebellum, dominant negative mutants of DDR1 block the elongation of granule neurones. DDR1 expression in human keratinocytes has been shown by Northern blotting, but up to now, the presence of DDR1 in skin has not been demonstrated by immunolocalization [54]. Here, sections through the mouse skin are presented that are stained with an antibody specific for DDR1 (Fig. 2). In the newborn mouse, strong expression of DDR1 is seen in the epidermis, being restricted to the basal layer of keratinocytes (Fig. 2A). Diffuse staining appears throughout the dermis and around the anlage of the hair follicle. In the skin of a 5 day old mouse, DDR1 expression is confined to the epidermis and the external root sheath of the hair (Fig. 2B).

Compared to other RTK, the juxtamembrane region of DDR1 or DDR2 is significantly longer (176 and 147 amino acids, respectively). The extended juxtamembrane region might have an autoinhibitory function in response to ligand binding, as it has been recently suggest for other RTK [46]. An autoinhibitory role of the juxtamembrane region would also provide a valid explanation for the protracted activation of DDR upon collagen engagement [52]. In all cell lines tested, DDR1 is partially processed into a 63 kDa membrane-anchored beta-subunit and a 54 kDa soluble, extracellular domain-containing alpha-subunit. The identity of the protease that is responsible for DDR1 shedding is not known. Thus far, five isoforms of the DDR1 protein have been characterized, that arise by alternative splicing [64]. The longest DDR1 transcript codes for the full-length 919 amino acid long receptor (c-isoform), whereas the a- or b-receptor isoforms lack 37 or 6 amino acids in the juxtamembrane or kinase domain, respectively [51]. The DDR1b protein is the predominant isoform expressed during embryogenesis, whereas the a-isoform is upregulated in certain human mammary carcinoma cell lines [65]. The relative expression ratio and the post-translational modification of the a- and b-isoform seem to be controlled by complex regulatory mechanisms. Overexpression of DDR1a and DDR1b in human embryonic kidney 293 cells revealed a differential glycosylation pattern of the two isoforms [51]. The alternatively spliced 37 amino acids insert in DDR1b contains the motif LLXNPXY, which can associate with the phosphotyrosine-binding domain of the ShcA adapter protein upon collagen-induced tyrosine phosphorylation [52]. Furthermore, the fibroblast-growth factor receptor substrate 2 (FRS2) binds to a chimeric molecule containing the juxtamembrane region of DDR1a [66]. The DDR1 isoforms d and e are truncated variants that either lack the entire kinase region or parts of the juxtamembrane region and the ATP binding site [64]. No splice variants for DDR2 have been identified so far.

Mice lacking DDR1 are smaller than control littermates, and mutant females show multiple reproductive defects [67]. The majority of the females are unable to give birth because developing blastocysts do not implant. Successfully reproducing females are unable to nourish their litters because the mammary gland epithelium fails to secret milk. Proliferation, morphology and collagen deposition are altered in the mutant mammary gland epithelium. Upon injury of the carotid artery, DDR1-null mice show reduced level of neointima formation and collagen deposition compared to control mice [68]. Mice with a deletion of DDR2 suffer from dwarfism as well, which is caused by a reduced proliferation rate of chondrocytes during puberty [69]. Results from mice lacking both DDR have not yet been obtained. Interestingly, mice lacking aortic carboxypeptidase-like protein, which has two discoidin homology domains and also binds to ECM, show impaired wound healing as well [70].

Evolutionary conserved collagen-receptors

Before the completion of the human genome sequencing program, the worm (Caenorhabditis elegans) and fly (Drosophila melanogaster) genomes were sequenced. Interestingly, integrins are present in both invertebrates, but with strikingly different diversity [71]. Whereas worms happily live with only one alphabeta integrin, flies have genes coding for five alpha and two beta subunits. Others found 7 alpha and 2 beta subunits in flies and 4 alpha and 2 beta subunits in worms [2]. In contrast, the number of DDR related genes persisted throughout evolution: flies and worms have also two each. The DDR-related gene CG9490 in the Drosophila genome has tentatively been named dDDR1 and CG9488 named dDDR2. The genes C25F.6 and F11D5.3 from C. elegans have been assigned as wDDR1 and wDDR2, respectively. Although both invertebrates show an abundant number of genes with homology to collagens, we are still awaiting experimental data showing that invertebrate integrins and DDR are functional collagen receptors [72].

Downstream targets of collagen-receptor signaling

Activation of DDR1 or DDR2 by collagen results in sustained intracellular phosphorylation [52, 53]. Several cytoplasmic tyrosine residues have been mapped to be phosphorylated and to serve as potential substrate binding sites (W. Vogel, unpublished results). For example, tyrosine-513 in the alternatively spliced insert of DDR1b allows binding of the adaptor protein ShcA with nanomolar affinity [52]. Although ShcA is not a substrate of DDR1b and does not induce Erk kinase activation, it might have other functions by regulating the architecture of the cytoskeleton. Similarly, the function of FRS2 binding to the juxtamembrane region of DDR1a has not been comprehensively analyzed [66]. Potentially, DDR1 expression is regulated during the cell cycle as evidence has been presented that overexpression of p53 in osteosarcoma cells induces DDR1 expression [73]. Recently published data imply that the Wnt-5a pathway may overlap with DDR1 signaling [74]. For DDR2, no direct binding partners have been identified so far. However, prolonged activation of DDR2 in a fibrosarcoma cell line induces MMP1 expression suggesting a potential regulatory mechanism between fibrillar collagen binding and MMP1-mediated proteolysis [52]. Currently, there is a need to search for additional DDR targets, for example by interaction screening or mass spectroscopy.

The expression and action of integrins are predominantly localized to the focal adhesion complex as originally demonstrated in fibroblasts. Focal adhesion complexes are the structural connection between the ECM and the cytoskeleton. Here, transmembrane signaling is mediated by integrins that cluster with dozens of cytoskeletal proteins and signaling molecules. Interestingly, integrins not only convey signals from the ECM into the cell (outside-in), but also react to cytoplasmic alterations by altering their capability to adhere to the ECM (inside-out).

After collagen-activation of integrins, the incoming signal is translated into increased tyrosine phosphorylation. Unlike the DDR, which have an intrinsic catalytic activity, integrins recruit cytoplasmic tyrosine kinases, in particular focal adhesion kinase (FAK), a 125 kDa multi-domain protein. The beta1 integrin cytoplasmic tail interacts with paxillin and talin, which both complex FAK [75]. The detailed mechanism, how FAK interacts with these proteins and becomes activated in response to integrin ligation, is not fully understood. It is clear that the autophosphorylation of FAK at several tyrosine residues enables the kinase to phosphorylate a number of other, mainly cytoskeletal proteins, including paxillin, talin, alpha-actinin, tensin, zyxin, VASP and vinculin. These signaling molecules then recruit ShcA, p130^Cas, Crk, PI3-kinase, Grb2 and tyrosine kinases like Src to the focal adhesion complex [76, 77]. In this signaling complex, Grb2 is of particular importance, since the small adaptor protein triggers the Ras pathway upon integrin engagement. Activated Ras initiates a cascade of Ser/Thr kinases, including Erk and Jnk, that ultimately initiate cell proliferation. Further downstream of the FAK signaling cascade are members of the Rho family of small GTPase proteins, that coordinate the plasticity of the actin cytoskeleton [78]. Vinculin and paxillin are scaffolding proteins, that provide multiple SH2 and SH3-domain depended interaction sites as well as connecting to microtubules and other cytosceletal structures [79]. For alpha2beta1, the ability to phosphorylate p38 MAPK and to enter the S-phase of cell cycle has been pinpointed to a short conserved stretch of amino acids in the cytoplasmic domain of the alpha subunit [80].

Depending on the cell type studied, different pathways have been found to be mainly activated by collagen-binding integrins. In colon cancer-derived epithelial cells stimulated with type IV collagen, Erk activation and hence cell proliferation are mediated by FAK, without the involvement of ShcA or p130^Cas [81]. In contrast, smooth muscle cells lining the normal arterial wall do not proliferate in response to native collagen. In these cells, only collagenase-degraded collagen simulates FAK activation and induces cleavage of FAK, paxillin and talin [82]. It has been suggested that partial denaturation of collagen unmasks cryptic RGD sequences that in turn modify the affinity towards integrins. It is clear that the cleavage of FAK is executed by calpain and that it results in a disassembly of focal adhesion complexes [83]. The relevance of FAK cleavage and hence its turnover for the ability of cellular migration is supported by the observation that fibroblasts from FAK-null animals show an increased number of focal adhesions but a reduced migration on ECM [84]. In platelets, yet another regulation of collagen-signaling pathways is observed. Blocking of the predominantly expressed integrins for collagen (alpha2beta1) or fibronectin (alphaIIbeta3) does not inhibit FAK activation [85]. Instead, the collagen receptor glycoprotein VI seems to mediate FAK phosphorylation by a protein kinase C-dependent pathway. In many different cell lines, alpha1beta1 stimulation induces collagen type I synthesis [86]. Patients with systemic sclerosis are characterized by excessive collagen production in the skin. Whether fibroblasts derived from these patients express reduced levels of alpha1beta1 integrin on their surface, is discussed by two conflicting reports [87, 88]. Presumably, the answer will come from the direct analysis of the tissues involved.

The third type of collagen-receptors, glycoprotein VI (GPVI), is a member of the IG superfamily and is constitutively associated with the Fc-gamma chain [89]. Expression of GPVI is only found in platelets and their precursor cells. Surprisingly, during the differentiation of megakaryocytes, targets commonly found in the integrin pathway, like p38 or PLC-gamma, are activated as well [90, 91].

Collagen-receptors and matrix metalloproteinases: Yin and Yang of the ECM

The synthesis and activation of the 26 known MMP are very tightly controlled in the tissue. Most of the MMP are released by cells as proenzymes and later activated in the extracellular space, mainly by serin proteases. Additionally, MMP are often trapped in a deactivating complex with tissue inhibitors of metalloproteinases (TIMP). The induction of MMP expression upon collagen stimulation has been reported from several different cell lines, including keratinocytes, smooth muscle and osteoprogenitor cells [92]. Major functions of the alpha2beta1 receptor include the induction of MMP1, MMP13 and MT1-MMP, as well as activation of proMMP2 [18, 93-95]. In human fibroblasts cultivated in floating collagen gels, the induction of MMP1 is mediated by PKC-zeta and p38 MAPK [96-98]. Interestingly, collagen-induced ligation of alpha2beta1 was also reported to upregulate collagen levels, thereby antagonizing the alpha1beta1 function [99].

More importantly, the formation of a direct complex between alpha2beta1 and pro-MMP1 has been recently shown in keratinocytes plated on native type I collagen [100]. In this complex, the collagen-binding epitope has been mapped to the hemopexin domain of pro-MMP1. While binding to the I domain of the alpha2 subunit, pro-MMP1 physically competes with binding of collagen to the same site [101]. Based on the realization of a direct MMP/integrin interaction, the following chain of events can be postulated. (i) Tissue injury exposes epithelial cells to proximally located fibrillar collagen and triggers integrin ligation. (ii) The newly formed adhesive contact between collagen and integrins induces transcriptional upregulation of MMP1 in the cell. (iii) By a so far unknown mechanism, pro-MMP1 is locally secreted and binds to a subset of I domains in the alpha2beta1 integrin. (iv) Formation of this ternary complex converts pro-MMP1 into its active form. This activation occurs either by another protease or by a conformational reorganization of the catalytic domain. Activated MMP1 then cleaves type I collagen at a single site. (v) Cleaved collagen rapidly denatures, which reduces its integrin-binding affinity. (vi) Consequently, the focal adhesion complex disassembles and allows the reorganization of the cytoskeleton. (vii) The cell can then move forward and form novel cell/matrix contacts. This chain of events is supported from data studying migration of VSMC on collagen, where MMP1 activity was localized only to discrete zones underneath the leading edge and the cell tail [102]. However, further details controlling the speed and regulation of cell migration need to be fully characterized.

Localization of MMP to cell surface receptors that do not bind collagen has been shown for MMP2 binding to alphaVbeta3, MMP9 binding to CD44 and MMP7 binding to heparan sulfate proteoglycans [103-105]. Furthermore, recent results suggest that MT1-MMP mediated cellular invasion of ovarian cancer cells through a collagen matrix depends on alpha2beta1 ligation and pro-MMP2 processing [106]. In these cells, MT1-MMP seems to cluster with several integrins and TIMP2 at cellular "hot spots", which are defined membrane protrusions named invadopodia.

Collagen receptors in wound healing, scarring and keloid formation

Now, which are the matrix receptors, collagens and MMP that are truly essential for wound healing? In vitro data demonstrate that downregulation of collagen synthesis by dermal fibroblasts is dependent upon alpha1beta1 function, but not alpha2beta1 [93, 107]. Studies with primary human keratinocytes suggest that integrin mediated migration on dermal type I collagen is initiated only 3-6 hrs after wounding [108]. Concomitantly, expression and tyrosine phosphorylation of FAK have been reported to peak 12 hrs after injury and to correlate with maximal cell migration [109]. Others reported a more gradual increase of FAK expression over a period of several days until re-epithelialization is completed [110]. Whole skin explants showed that FAK activation is not restricted to the wound margins, but is also observed some cell diameters away from the migrating cells [111]. In cells at the wound margin, FAK activation by collagen ligation of alpha2beta1 induces laminin5 deposition, which triggers alpha3beta1 and alpha6beta4-mediated FAK signaling in cells more distant from the wound edge [112]. External regulators, particularly transforming growth factor-beta, also modulate keratinocyte migration by inducing collagen and integrin gene transcription [113, 114]. Following wound closure, the proliferation of dermal fibroblasts appears to be terminated by apoptosis [115]. This process is also collagen type I dependent and can be inhibited by alpha1beta1 and alpha2beta1 blocking antibodies [116]. In contrast to many epithelial cells, in which cell death can be induced by detachment from the substratum, termed anoikis, apoptosis in dermal fibroblasts seems to be mediated by active collagen-receptor signaling.

In vivo, wound healing has been analyzed in an explant model by transplanting human skin onto severe combined immunodeficient mice [117]. Additionally, knockout mice turned out to be ideal to prove the involvement of a particular gene product in wound healing. In a commonly used model, excisional wounds are placed on the back of the animal, and wound closure is monitored for up to 14 days [118]. Dermal wounds applied to DDR1 knockout mice healed as control wounds suggesting that the presence of DDR1 is not essential for wound healing (unpublished observation, B. Eckes and W. Vogel). However, DDR1 is linked to excessive collagen deposition during keloid formation. Comparing normal dermal fibroblasts with primary human keloid cells, increased expression and tyrosine phosphorylation of DDR1 was found [119]. In this study, the induction of DDR1 parallels with an overall increase in cellular tyrosine phosphorylation and EGF receptor overexpression. Concomitantly, the adaptor protein ShcA, a common target for DDR1 and EGF receptor is upregulated. In DDR2 knockout mice, the number of proliferating cells after wounding is drastically reduced [69]. Furthermore, DDR2-minus dermal fibroblasts cultured in vitro grow significantly slower than wild type cells.

From the data accumulated so far, it is tempting to propose integrins and DDR as therapeutical targets aiming for accelerated wound healing and reduced keloid formation. One may speculate on the availability of specific small molecule compounds, which either interfere with the binding of collagen to integrins/DDR or which act as specific inhibitors of the FAK or DDR tyrosine kinase activity. Potential benefits would be accelerated wound closure or increased quality of dermal scars. To reach these goals, we clearly need a much deeper understanding of the mechanisms of collagen-receptor signaling.

CONCLUSION

Acknowledgements

The author would like to thank F. Alves, C. Brakebusch, J. Eble, B. Eckes and S.V. Jassal for critically commenting the manuscript. This work was supported by grants from DFG (V663/2-1) and BMBF.

Article accepted on 11/8/01

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