Correction: Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0068301.].

Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

The dual leucine zipper kinase DLK induces ß-cell apoptosis by inhibiting the transcriptional activity conferred by the ß-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to ß-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1ß, known prediabetic signals. In the present study, the regulation of DLK in ß-cells by these cytokines was investigated. Both, TNFα and IL-1ß induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the ß-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of ß-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

Renal cells express different forms of vimentin: the independent expression alteration of these forms is important in cell resistance to osmotic stress and apoptosis.

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.

Alport syndrome--insights from basic and clinical research.

In 1927, Arthur C. Alport first published his description of a triad of symptoms in a family with hereditary congenital haemorrhagic nephritis, deafness and ocular changes. A few years after his death, this group of symptoms was renamed Alport syndrome. To this day, Alport syndrome still inevitably leads to end-stage renal disease and the need for renal replacement therapy, starting in young adulthood. During the past two decades, research into this rare disease has focused on the effects of mutations in collagen type IV and the role of changes in podocytes and the glomerular basement membrane that lead to early kidney fibrosis. Animal models of Alport syndrome also demonstrate the pathogenetic importance of interactions between podocytes and the extracellular matrix. Such models might also help researchers to answer basic questions about podocyte function and the development of fibrosis, and to develop new therapeutic approaches that might be of use in other kidney diseases. In this Review, we discuss the latest basic and clinical research on Alport syndrome, focusing on the roles of podocyte pathology and the extracellular matrix. We also highlight early diagnosis and treatment options for young patients with this disorder.

New insights into cartilage repair - the role of migratory progenitor cells in osteoarthritis.

Osteoarthritis is one of the most common musculo-skeletal diseases with a complex patholoy and a strong impact on cell biology, differentiation and migration behavior of mesenchymal stem cell-derived progenitor cells. In this review, we elucidate the influence of the pathologically altered extracellular matrix on progenitor cell behavior. Moreover, we discuss the modulation of progenitor cells especially of previously characterized chondrogenic progenitor cells (Koelling et al., 2009) in situ to enhance their regeneration potential. These options comprise the application of growth factors like fibroblast growth factor-2, a Runx-2 knock down and a contemporary anti-inflammatory therapy. This supports endogenous regeneration on behalf of the diseased osteoarthritic cartilage, which otherwise results mainly in an insufficient fibro-cartilaginous repair tissue. Furthermore, new results indicate a role of pericytes in osteoarthritis for these repair attempts. We discuss the biological mechanisms potentially leading to new therapeutic options in osteoarthritis to enhance regeneration in situ.

Integrin α2-deficient mice provide insights into specific functions of collagen receptors in the kidney.

BACKGROUND: Integrins are important cellular receptors for collagens. Within the glomerulus, podocytes regulate the integrity of the glomerular basement membrane (GBM) by sensing the presence of collagen and regulating collagen IV synthesis. The present study evaluates the role of integrin α2 (ITGA2) in cell-matrix interaction. METHODS AND RESULTS: ITGA2-deficient mice had normal renal function but moderate proteinuria and enhanced glomerular and tubulointerstitial matrix deposition. Electron microscopy demonstrated irregular podocyte-matrix interaction, causing pathological protrusions towards the urinary (podocyte) side of the GBM. These characteristic subepithelial bulges mimic the renal phenotype of mice, which are deficient in another collagen receptor, discoidin domain receptor (DDR)1. Using immunogold staining, ITGA2 expression was found to localize to the basolateral site of the podocyte foot processes. ITGA2-deficient mice overexpressed transforming growth factor (TGF)ß and connective tissue growth factor (CTGF) compared with wild-type mice. Using in situ hybridization, tubular cells were found to be the primary site of TGFß synthesis and podocytes the source of CTGF in ITGA2-deficient mice. CONCLUSION: These findings support our hypothesis that both these collagen receptors (ITGA2 and DDR1) play a similar role within the kidney. Further, cell-matrix interaction via collagen receptors seems to be crucial for maintenance of normal GBM architecture and function. Targeting collagen receptors such as ITGA2 might be a new form of treatment for progressive fibrotic diseases.

Basement membrane components are key players in specialized extracellular matrices.

More than three decades ago, basement membranes (BMs) were described as membrane-like structures capable of isolating a cell from and connecting a cell to its environment. Since this time, it has been revealed that BMs are specialized extracellular matrices (sECMs) with unique components that support important functions including differentiation, proliferation, migration, and chemotaxis of cells during development. The composition of these sECM is as unique as the tissues to which they are localized, opening the possibility that such matrices can fulfill distinct functions. Changes in BM composition play significant roles in facilitating the development of various diseases. Furthermore, tissues have to provide sECM for their stem cells during development and for their adult life. Here, we briefly review the latest research on these unique sECM and their components with a special emphasis on embryonic and adult stem cells and their niches.

Loss of collagen-receptor DDR1 delays renal fibrosis in hereditary type IV collagen disease.

Alport syndrome is a hereditary type IV collagen disease leading to progressive renal fibrosis, hearing loss and ocular changes. End stage renal failure usually develops during adolescence. COL4A3-/- mice serve as an animal model for progressive renal scarring in Alport syndrome. The present study evaluates the role of Discoidin Domain Receptor 1 (DDR1) in cell-matrix interaction involved in pathogenesis of Alport syndrome including renal inflammation and fibrosis. DDR1/COL4A3 Double-knockouts were compared to COL4A3-/- mice with 50% or 100% expression of DDR1, wildtype controls and to DDR1-/- COL4A3+/+ controls for over 6years. Double-knockouts lived 47% longer, mice with 50% DDR1 lived 29% longer and showed improved renal function (reduction in proteinuria and blood urea nitrogen) compared to animals with 100% DDR1 expression. Loss of DDR1 reduced proinflammatory, profibrotic cells via signaling of TGFbeta, CTGF, NFkappaB and IL-6 and decreased deposition of extracellular matrix. Immunogold-staining and in-situ hybridisation identified podocytes as major players in DDR1-mediated fibrosis and inflammation within the kidney. In summary, glomerular epithelial cells (podocytes) express DDR1. Loss of DDR1-expression in the kidney delayed renal fibrosis and inflammation in hereditary type IV collagen disease. This supports our hypothesis that podocyte-matrix interaction via collagen receptors plays an important part in progression of renal fibrosis in Alport disease. The blockade of collagen-receptor DDR1 might serve as an important new therapeutic concept in progressive fibrotic and inflammatory diseases in the future.

Botulinum toxin: A noninvasive option for the symptomatic treatment of salivary gland stenosis - a case report.

BACKGROUND: A man diagnosed with Stensen's duct stenosis exhibited recurrent parotid swelling, invariably during meals. Previous parotid duct dilations and percutaneous radiotherapy were ineffective. Botulinum toxin (BTX) injections were injected into the affected gland to regulate salivary flow and reduce parotid swelling. METHODS: BTX (22.5 units) was injected into the affected gland. A second treatment with 30 units BTX was carried out 7 weeks later. Two further injections followed after 4 months, respectively. The results were scored by the patient and evaluated in an examination. RESULTS: The patient reported the disappearance of parotid swelling after 2 weeks of injections. This effect was maintained for 5 weeks after the first treatment and for 4 months after the following 2 treatments. There were no side effects. CONCLUSION: Here we introduce BTX as a therapeutic option for the treatment of salivary duct stenosis when other therapies are ineffective and before opting for gland extirpation.

Tissue distribution of perlecan domains III and V during embryonic and fetal human development.

A major component of basement membranes (BMs) is perlecan, a five-domain heparan sulphate proteoglycan. During murine embryogenesis, nearly all BMs of mesenchymal origin express perlecan, and it is believed to participate in the supramolecular assembly of BMs. However, the distribution of perlecan in human embryonic and fetal tissues is widely unknown, except for cartilage anlagen of developing extremities and the fetal spine. Clinical syndromes, caused by perlecan-associated mutations or gene-defects, suggest its multifunctional involvement during human development. Here we reveal the immunohistochemistry of perlecan domains III and V during human development from gestational weeks (gw) 6 to 12 in basement membrane zones (BMZs) of the developing brain, nervous system, blood vessels, skin, lung, heart, kidney, liver, intestine and skeletal system. Interestingly, a difference in the distribution of the two perlecan domains was found in the endoneurium of ganglia. Domain III is strongly present from gw 6 onwards, while domain V shows attenuated expression at this stage and has been detected abundantly only from gw 8 onwards, possibly indicating vascularization of the endoneurium during this early stage. We found perlecan to be present particularly at those stages of human development where epithelial-mesenchymal interactions occur.

Migratory chondrogenic progenitor cells from repair tissue during the later stages of human osteoarthritis.

The regeneration of diseased hyaline cartilage continues to be a great challenge, mainly because degeneration--caused either by major injury or by age-related processes--can overextend the tissue's self-renewal capacity. We show that repair tissue from human articular cartilage during the late stages of osteoarthritis harbors a unique progenitor cell population, termed chondrogenic progenitor cells (CPCs). These exhibit stem cell characteristics such as clonogenicity, multipotency, and migratory activity. The isolated CPCs, which exhibit a high chondrogenic potential, were shown to populate diseased tissue ex vivo. Moreover, downregulation of the osteogenic transcription factor runx-2 enhanced the expression of the chondrogenic transcription factor sox-9. This, in turn, increased the matrix synthesis potential of the CPCs without altering their migratory capacity. Our results offer new insights into the biology of progenitor cells in the context of diseased cartilage tissue. Our work may be relevant in the development of novel therapeutics for the later stages of osteoarthritis.

Nidogen-1 and nidogen-2 in healthy human cartilage and in late-stage osteoarthritis cartilage.

OBJECTIVE: To investigate the presence and function of nidogen-1 and nidogen-2 in healthy human cartilage and in late-stage osteoarthritis (OA) cartilage. METHODS: The location and quantity of nidogen-1 and nidogen-2 protein and messenger RNA were determined in cartilage tissue obtained from healthy donors and from patients with late-stage knee OA. Samples were analyzed by immunohistochemistry, in situ hybridization, and real-time reverse transcription-polymerase chain reaction. Adhesion and inhibition assays, a pre-embedding method, fluorescence-activated cell sorting, and ultrastructural investigations with integrins were also carried out. RESULTS: Developing tissue from human embryos showed strong staining for both nidogens in condensed mesenchyme and in rib anlagen. Homogeneous staining for nidogen-1 was observed in the extracellular matrix of healthy articular cartilage, whereas nidogen-2 was localized pericellularly. In late-stage OA cartilage, expression of nidogen-1 was decreased pericellularly around diseased chondrocytes, whereas nidogen-2 was increased. However, both nidogens had strongly increased levels around elongated chondrocytes, especially in areas of deep surface fissures. In vitro, both nidogens functioned as adhesion proteins for cells from the OA defect. In vivo, colocalizations with integrins alphav and beta1 as well as internalization of nidogens by chondrocytes in vitro were observed. CONCLUSION: Nidogens are involved in human limb development. They occur in healthy articular cartilage and show increased expression, primarily around elongated chondrocytes, in OA cartilage. Therefore, the activities of nidogens might be a sign of cartilage regeneration in late-stage OA. Furthermore, the adhesive character of nidogens, specifically as adhesion proteins for chondrocytes from late-stage OA, as well as the enhanced chondrocyte-nidogen interaction in OA indicate that both proteins play a key role in the pathogenesis of OA and either could be applied as a diagnostic marker.

Collagen IX in weight-bearing areas of human articular cartilage in late stages of osteoarthritis.

AIM: To determine if the amounts of collagen IX mRNA and protein are higher in osteoarthritic cartilage from weight-bearing areas of the knee joint compared to non-weight bearing areas in patients with stage IV osteoarthritis (OA). METHODS: Normal and OA cartilage samples were obtained from 15 patients undergoing total knee replacement or necropsies. mRNA was measured by real time RT-PCR and proteins were detected by Western blot and localized at the light and ultrastructural level. RESULTS: Collagen IX was found throughout all cartilage layers in healthy and OA tissue. Cells deposited collagen IX in the pericellular and interterritorial matrix and a 66% higher amount of collagen IX was detected in the pericellular matrix of the weight-bearing areas adjacent to the main defect in comparison to the macroscopically intact areas. This is in line with a 3.72 times higher amount of the respective mRNA. CONCLUSION: The increased levels of collagen IX protein and its mRNAs found in the weight-bearing areas adjacent to the main cartilage defect might reflect an attempt on the part of the diseased cartilage tissue to stabilize and protect the remaining matrix of late-stage osteoarthritic cartilage from further destruction.