Dual role of laminin­511 in regulating melanocyte migration and differentiation.

Laminins are the major basement membrane (BM) components and are heterotrimers composed of an α, a ß and a γ chain. In skin, laminins are present in basement membranes surrounding vascular structures, nerves, adipose tissue and in the specialized junctional BM between the epidermis and dermis. The main laminin isoforms in the dermo-epidermal BM are laminin­332, laminin­511 and laminin­211, the latter being restricted to hair follicles (HFs). The laminin γ1 chain is the most abundant γ chain; its global ablation in mice leads to early embryonic lethality at E5.5. To elucidate the cellular function of the γ1 chain in skin, we generated mice with keratinocyte-specific deletion of this chain (Lamc1EKO) by using the keratin (K)14-Cre/loxP system. These mice showed delayed coat pigmentation despite normal melanocyte counts in the skin. However, levels of differentiation-specific melanocyte enzymes TRP­1, TRP­2 and tyrosinase were reduced in Lamc1EKO mice, and melanocytes failed to migrate to their differentiation niche in HFs and accumulated in the IFE. These results suggested that the pigmentation defect results from impaired melanocyte migration. The impaired migratory capacity of melanocytes is due to the altered composition of laminins in the BM of Lamc1EKO mice: Loss of keratinocyte-derived pro-migratory laminin­511 is not compensated by ectopically deposited fibroblast-derived laminin­211. Furthermore, contact of melanocytes with recombinant laminin­511, but not with laminin­211, induces the expression of the chemokine receptor CXCR4 on melanocytes, needed for SDF­1 (stromal cell­derived factor­1)-mediated migration into HFs. We here demonstrate that laminin­511 controls the differentiation of melanocytes by regulating their migration from the epidermis into HFs and by activating CXCR4 expression on melanocytes required for their recruitment into HFs in an SDF­1-dependent manner.

Laminin α5 in the keratinocyte basement membrane is required for epidermal-dermal intercommunication.

Laminin α5 is broadly expressed in the epidermal basement membrane (BM) of mature mice and its elimination at this site (Lama5Ker5 mouse) results in hyperproliferation of basal keratinocytes and a delay in hair follicle development, which correlated with upregulation of the dermally-derived laminin α2 and laminin α4 chains in the epidermal BM and of tenascin-C subjacent to the BM. In vitro studies revealed laminin 511 to be strongly adhesive for primary keratinocytes and that loss of laminin α5 does not result in cell autonomous defects in proliferation. Flow cytometry reveals that the loss of laminin α5 resulted in increased numbers of CD45+, CD4+ and CD11b+ immune cells in the skin, which temporo-spatial analyses revealed were detectable only subsequent to the loss of laminin α5 and the appearance of the hyperproliferative keratinocyte phenotype. These findings indicate that immune cell changes are the consequence and not the cause of keratinocyte hyperproliferation. Loss of laminin α5 in the epidermal BM was also associated with changes in the expression of several dermally-derived growth factors involved in keratinocyte proliferation and hair follicle development in adult but not new born Lama5Ker5 skin, including KGF, EGF and KGF-2. In situ binding of FGF-receptor-2α (IIIb)-Fc chimera (FGFR2IIIb) to mouse skin sections revealed decoration of several BMs, including the epidermal BM, which was absent in Lama5Ker5 skin. This indicates reduced levels of FGFR2IIIb ligands, which include KGF and KGF-2, in the epidermal BM of adult Lama5Ker5 skin. Our data suggest an initial inhibitory effect of laminin α5 on basal keratinocyte proliferation and migration, which is exacerbated by subsequent changes in growth factor expression by epidermal and dermal cells, implicating laminin α5 in epidermal-dermal intercommunication.

Deletion of the epidermis derived laminin γ1 chain leads to defects in the regulation of late hair morphogenesis.

Laminins are the most abundant non-collagenous basement membrane (BM) components, composed of an α, ß and γ chain. The laminin γ1 chain, encoded by LAMC1, is the most abundant γ chain. The main laminin isoforms in the dermo-epidermal junction (DEJ) are laminin-332, laminin-511 and laminin-211, the latter being restricted to the lower part of hair follicles (HFs). Complete deletion of LAMC1 results in lethality around embryonic day 5.5. To study the function of laminin γ1 containing isoforms in skin development and maturation after birth, we generated mice lacking LAMC1 expression in basal keratinocytes (LAMC1EKO) using the keratin 14 (K14) Cre/loxP system. This deletion resulted in loss of keratinocyte derived laminin-511 and in deposition of fibroblast derived laminin-211 throughout the whole DEJ. The DEJ in areas between hemidesmosomes was thickened, whereas hemidesmosome morphology was normal. Most strikingly, LAMC1EKO mice showed delayed HF morphogenesis accompanied by reduced proliferation of hair matrix cells and impaired differentiation of hair shafts (HS). However, this deletion did not interfere with early HF development, since placode numbers and embryonic hair germ formation were not affected. Microarray analysis of skin revealed down regulation of mainly different hair keratins. This is due to reduced expression of transcription factors such as HoxC13, FoxN1, FoxQ1 and Msx2, known to regulate expression of hair keratins. While the role of laminin-511 in signaling during early hair germ formation and elongation phase has been described, we here demonstrate that epidermal laminin-511 is also a key regulator for later hair development and HS differentiation.

Tetanus toxin entry. Nidogens are therapeutic targets for the prevention of tetanus.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in nonvaccinated areas. TeNT targets the neuromuscular junction (NMJ) with high affinity, yet the nature of the TeNT receptor complex remains unknown. Here, we show that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Inhibition of the TeNT-nidogen interaction by using small nidogen-derived peptides or genetic ablation of nidogens prevented the binding of TeNT to neurons and protected mice from TeNT-induced spastic paralysis. Our findings demonstrate the direct involvement of an extracellular matrix protein as a receptor for TeNT at the NMJ, paving the way for the development of therapeutics for the prevention of tetanus by targeting this protein-protein interaction.

Anti-angiogenic effect of the basement membrane protein nidogen-1 in a mouse model of choroidal neovascularization.

In patients with age-related macular degeneration disruption of the integrity of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM), precedes choroidal neovascularization (CNV). We investigated the role of the basement membrane (BM) proteins nidogen-1 and nidogen-2 for the development of experimental CNV. Laser-induced CNV was studied in Nid1(-/-) and Nid2(-/-) mice and wild type (WT) controls by fluorescein angiography, by immune histochemistry of flat-mounts or paraffin sections to analyze expression pattern of nidogen-1 and -2 and nidogen binding BM proteins, and by western blotting. The influence of VEGF and bFGF on the mRNA expression of nidogen-1 was studied in vitro. Nidogen-1 protein is present in the BM of the inner limiting membrane (ILM), the retinal capillaries, and the choroid/sclera and CNV. Nidogen-2 protein is also found in these BMs but with a weaker expression in the ILM. In the retina the absence of nidogen-1 does not influence the expression of nidogen-2 and vice versa and does not influence the expression of the BM components collagen IV, laminin γ1, and perlecan. In Nid1(-/-) mice, CNV lesions showed increased vessel leakage during angiography and the CNV area was larger than in WT or nidogen-2 deficient mice. Laser treatment led to up-regulation of nidogen-1 protein expression in the sclera/choroid of nidogen-2 deficient or WT mice. The treatment of HUVECs with VEGF leads to a reduced expression of nidogen-1 mRNA whereas its expression remained unchanged in RPE cells. In conclusion, nidogen-1 produced by the endothelial cells acts as a factor to help stabilizing the BM, thus preventing the sprouting of new vessels or the infiltration of endothelial cells. In this sense nidogen-1 is essential to provide an anti-angiogenic environment of differentiated vessels.

Skin basement membrane: the foundation of epidermal integrity--BM functions and diverse roles of bridging molecules nidogen and perlecan.

The epidermis functions in skin as first defense line or barrier against environmental impacts, resting on extracellular matrix (ECM) of the dermis underneath. Both compartments are connected by the basement membrane (BM), composed of a set of distinct glycoproteins and proteoglycans. Herein we are reviewing molecular aspects of BM structure, composition, and function regarding not only (i) the dermoepidermal interface but also (ii) the resident microvasculature, primarily focusing on the per se nonscaffold forming components perlecan and nidogen-1 and nidogen-2. Depletion or functional deficiencies of any BM component are lethal at some stage of development or around birth, though BM defects vary between organs and tissues. Lethality problems were overcome by developmental stage- and skin-specific gene targeting or by cell grafting and organotypic (3D) cocultures of normal or defective cells, which allows recapitulating BM formation de novo. Thus, evidence is accumulating that BM assembly and turnover rely on mechanical properties and composition of the adjacent ECM and the dynamics of molecular assembly, including further "minor" local components, nidogens largely functioning as catalysts or molecular adaptors and perlecan as bridging stabilizer. Collectively, orchestration of BM assembly, remodeling, and the role of individual players herein are determined by the developmental, tissue-specific, or functional context.

Loss of epidermal MMP-14 expression interferes with angiogenesis but not with re-epithelialization.

Synthesis and activation of matrix metalloproteinases during wound healing are important for remodeling the extracellular matrix and modulating various cellular functions. The membrane-type 1 matrix metalloproteinase (MMP-14) has been shown to play a key role during these processes. To analyze the function of epidermal-derived MMP-14 during skin repair we generated mice lacking MMP-14 expression in the epidermis (MMP-14(ep-/-)). These mice displayed overall normal skin morphology and epidermal differentiation patterns. Wound repair in MMP-14(ep-/-) followed the same kinetics as in wild type mice (MMP-14(ep+/+)), and infiltration of neutrophils, leukocytes, and macrophages into the wound site was comparable. Microscopic analysis showed no altered re-epithelialization in the absence of epidermal MMP-14. Furthermore, epidermal differentiation at the end of the repair process and scar formation was normal. However, at day 14 post wounding, sustained angiogenesis was observed in MMP-14(ep-/-) mice in contrast to control mice. Interestingly, decreased levels of endostatin were detected in wound lysates of MMP-14(ep-/-) mice as well as in cultured keratinocytes. Taken together, these data indicate that MMP-14 expression in keratinocytes is dispensable for skin homeostasis and repair, but plays a crucial role in the epidermal-dermal crosstalk leading to modulation of vessel density.

Different domains in nidogen-1 and nidogen-2 drive basement membrane formation in skin organotypic cocultures.

Nidogen-1 and nidogen-2 are homologous proteins found in all basement membranes (BMs). They show comparable binding activities in vitro and partially redundant functions in vivo. Previously, we showed that in skin organotypic cocultures, BM formation was prevented in the absence of nidogens and that either nidogen was able to rescue this failure. We now dissected the two nidogens to identify the domains required for BM deposition. For that purpose, HaCaT cells were grown on collagen matrices containing nidogen-deficient, murine fibroblasts. After addition of nidogen-1 or nidogen-2 protein fragments comprising different binding domains, BM deposition was analyzed by immunofluorescence and electron microscopy. We could demonstrate that the rod-G3 domain of nidogen-2 was sufficient to achieve deposition of BM components at the epidermal-collagen interface. In contrast, for nidogen-1, both the G2 and G3 domains were required. Immunoblot analysis confirmed that all BM components were present in comparable amounts under all culture conditions. This finding demonstrates that nidogens, although homologous proteins, exert their effect on BM assembly through different binding domains, which may in turn result in alterations of BM structure and functions, thus providing an explanation for the phenotypical differences observed between nidogen-1 and -2 deficient mice.

Expression of platelet-derived growth factor-C and insulin-like growth factor I in hepatic stellate cells is inhibited by miR-29.

MicroRNAs are short noncoding, endogenous RNA species that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of mRNAs. Recently, it was shown that miR-29 inhibits expression of extracellular matrix proteins such as collagens, suggesting an antifibrotic function of miR-29. In the present study, we now investigated the role of miR-29 in profibrogenic growth factor expression as a further central mechanism of fibrosis. Screening of databases revealed putative miR-29 target sequences in the mRNA of platelet-derived growth factor (PDGF)-B, PDGF-B receptor, PDGF-C, vascular endothelial growth factor-A, and insulin-like growth factor (IGF)-I. To analyze miR-29 interaction with the predicted binding sites, we cloned the 3'-UTR sequences of the putative targets in fusion to the luciferase-reporter coding sequence. Functional miR-29 binding to PDGF-C and IGF-I mRNA sequences, but not to the corresponding mutants, was then proven by reporter assays. Hepatic stellate cells (HSC) that transdifferentiate into myofibroblasts, producing extracellular matrix proteins and profibrogenic growth factors, for example, the members of the PDGF family, are crucial for liver fibrosis. Myofibroblastic transition of primary HSC resulted in the loss of miR-29, but in a significant increase of PDGF-C and IGF-I. Compensation of reduced miR-29 levels by miR-29 overexpression in myofibroblastic HSC was followed by a definitive repression of IGF-I and PDGF-C synthesis. After experimental fibrosis, induced by bile-duct occlusion, miR-29 expression was shown to be reduced, but IGF-I and PDGF-C expression was upregulated, correlating inversely to the miR-29 pattern. Thus, we conclude that miR-29, downregulated during fibrosis, acts as an antifibrogenic mediator not only by targeting collagen biosynthesis, but also by interfering with profibrogenic cell communication via PDGF-C and IGF-I.

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier.

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice.

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.

Hepatocyte growth factor (HGF) inhibits collagen I and IV synthesis in hepatic stellate cells by miRNA-29 induction.

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-ß (TGF-ß) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-ß on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-ß, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-ß stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-ß stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-ß exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-ß stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-ß take part in the anti- or profibrogenic response of HSC, respectively.

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain.

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

The disintegrin-like and cysteine-rich domains of ADAM-9 mediate interactions between melanoma cells and fibroblasts.

A characteristic of malignant cells is their capacity to invade their surrounding and to metastasize to distant organs. During these processes, proteolytic activities of tumor and stromal cells modify the extracellular matrix to produce a microenvironment suitable for their growth and migration. In recent years the family of ADAM proteases has been ascribed important roles in these processes. ADAM-9 is expressed in human melanoma at the tumor-stroma border where direct or indirect interactions between tumor cells and fibroblasts occur. To analyze the role of ADAM-9 for the interaction between melanoma cells and stromal fibroblasts, we produced the recombinant disintegrin-like and cysteine-rich domain of ADAM-9 (DC-9). Melanoma cells and human fibroblasts adhered to immobilized DC-9 in a Mn(2+)-dependent fashion suggesting an integrin-mediated process. Inhibition studies showed that adhesion of fibroblasts was mediated by several ß1 integrin receptors independent of the RGD and ECD recognition motif. Furthermore, interaction of fibroblasts and high invasive melanoma cells with soluble recombinant DC-9 resulted in enhanced expression of MMP-1 and MMP-2. Silencing of ADAM-9 in melanoma cells significantly reduced cell adhesion to fibroblasts. Ablation of ADAM-9 in fibroblasts almost completely abolished these cellular interactions and melanoma cell invasion in vitro. In summary, these results suggest that ADAM-9 expression plays an important role in mediating cell-cell contacts between fibroblasts and melanoma cells and that these interactions contribute to proteolytic activities required during invasion of melanoma cells.

Combined paracrine and endocrine AAV9 mediated expression of hepatocyte growth factor for the treatment of renal fibrosis.

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1alpha1 (Col1A1), platelet-derived growth factor receptor-beta (PDGFR-beta), and alpha-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.

Cell-matrix interactions in dermal repair and scarring.

Regulation of cellular functions during dermal repair following injury is complex and critically dependent on the interaction of cells with the surrounding extracellular matrix (ECM). The ECM comprises various families of macromolecules that form the structural scaffold of the tissue, but also carry distinct biological activities. After injury to the skin, the defect is filled by a provisional matrix that is invaded by inflammatory cells, sprouting blood vessels and fibroblasts. In a later phase, the wound contracts, the tissue is replaced by mature connective tissue produced by activated fibroblasts, and a scar is formed. All cells involved communicate directly with the ECM by integrins and other matrix receptors. These transmit signals and induce adaptive responses to the environment by the embedded cells. The ECM or proteolytic fragments of individual ECM constituents exert defined biological activities influencing cell survival, differentiation of myofibroblasts, ECM synthesis and turnover, wound angiogenesis and scar remodeling. Extensive crosstalk exists between ECM and growth factors, and between growth factors and integrins. ECM-cell contact also enables direct transmission of mechanical tension, which then modulates many activities of all cellular players. Understanding this complex interplay is important to provide a basis for designing effective wound therapy and for strategic interference with mechanisms that have gone out of control in fibrotic conditions.

Basement membrane protein nidogen-1 shapes hippocampal synaptic plasticity and excitability.

The basement membrane (BM) is a specialized form of extracellular matrix (ECM) underlying epithelia and endothelia and surrounding many types of mesenchymal cells. Nidogen, along with collagen IV and laminin, is a major component of BMs. Although certain ECM proteins such as laminin or reelin influence neuronal function via interactions with cell-surface receptors such as integrins, behavioral neurological impairments due to deficits of BM components have been recognized only recently. Here, alterations in neuronal network function underlying these behavioral changes are revealed. Using nidogen-1 knockout mice, with or without additional heterozygous nidogen-2 knockout (NID1(-/-)/NID2(+/+) or NID1(-/-)/NID2(+/-)), we demonstrate that nidogen is essential for normal neuronal network excitability and plasticity. In nidogen-1 knockouts, seizurelike behavior occurs, and epileptiform spiking was seen in hippocampal in vivo EEG recordings. In vitro, hippocampal field potential recordings revealed that lack of nidogen-1, while not causing conspicuous morphological changes, led to the appearance of spontaneous and evoked epileptiform activity, significant increase of the input/output ratio of synaptically evoked responses in CA1 and dentate gyrus, as well as of paired pulse accentuation, and loss of perforant-path long-term synaptic potentiation. Nidogen-1 is thus essential for normal network excitability and plasticity.

Impaired wound healing in mice lacking the basement membrane protein nidogen 1.

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable alpha-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the beta1 and beta4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.

Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts.

Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney. The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGFbeta. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.

Basement membranes in skin: unique matrix structures with diverse functions?

The view of extracellular matrix (ECM) has evolved from a merely scaffolding and space filling tissue element to an interface actively controlling cellular activities and tissue functions. A highly specialized form of ECM is the basement membrane (BM), an ubiquitous sheet-like polymeric structure composed of a set of distinct glycoproteins and proteoglycans. In this review we are largely focusing on function and assembly of BM in skin (1) at the dermo-epidermal interface and (2) in the resident micro-vasculature. The role of the non-polymeric components perlecan and particularly nidogen is exemplified by reviewing experiments based on genetic approaches and adequate experimental skin models in vivo and in vitro. While in mice total deficiency of one of these components is eventually developmentally lethal, the severity of the defects varies drastically between tissues and also the skin models recapitulating BM formation in vitro. There is accumulating evidence that this relies on the mechanical properties, the molecular composition of the BM, the adjacent ECM or connective tissue, the dynamics of molecular assembly, and 'minor' tissue-specific modifier or adapter components. Though the role of nidogen or perlecan is still remaining a controversial issue, the statements 'being essential for BM/or not' should be consequently referred to the developmental, tissue, and functional (e.g., repair) context.