Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (≥50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1a mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-␤-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-␣-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.

AimsType 2 diabetes mellitus (DM) leads to cardiac dysfunction irrespective of hypertension and coronary artery disease; this is called diabetic cardiomyopathy. Here, we investigated the severity of diabetic cardiomyopathy and myocardial remodelling in aged Zucker diabetic fatty (ZDF) rats. Methods and resultsBody weight, blood glucose and glycated haemoglobin (Hb A1c ) levels, and urinary albumin excretion were monitored regularly in ZDF rats (n ¼ 19) and control littermates (n ¼ 19) up to age 45 weeks. ZDF rats were severely diabetic during the entire study period and demonstrated decreased body and heart weights at sacrifice. Left ventricular (LV) function was determined using magnetic resonance imaging (MRI) at age 44 weeks and revealed similar LV ejection fraction and cardiac output index in control and ZDF rats, indicating preserved systolic function. LV pressure characteristics assessed at age 45 weeks showed significant, but mild elevations of LV end-diastolic pressure (+45%) and relaxation time constant Tau (+54%) in ZDF rats, indicating diastolic dysfunction. Histological analyses revealed a significantly increased LV collagen content (+50%), but no cardiomyocyte hypertrophy in ZDF rats. ConclusionThe present study clearly shows that long term, severe DM in 45-week-old ZDF rats resulted in relatively mild impairment of diastolic LV function, whereas systolic function was well preserved. These data do not support the notion that diabetes per se is a critical factor in the induction of a clinically relevant degree of cardiac dysfunction. Co-morbidities such as hypertension and coronary artery disease probably have larger impacts on myocardial function in diabetic individuals.--

Our studies clearly demonstrate that marked differences exist between individual NSAIDs with respect to their ability to modulate the imbalance between proteases and inhibitors during OA and RA, suggesting that the respective modes of action are independent of the inhibition of cyclooxygenases. Due to their co-regulation of MMPs and TIMP(s) glucocorticoids should be carefully studied for their overall effect on ECM proteolysis.

In this study we analyzed the proteolytic activity of MMP-19 and its impact on keratinocyte migration. In the HaCaT keratinocyte cell line overexpressing wild-type MMP-19 (HaCaT-WT), transmigration through fibrin and type IV collagen matrices was significantly increased compared to cells harboring a catalytically inactive mutant (HaCaT-EA). Studying the expression of MMP-19 in early stages of squamous cell cancer (SCC), we found co-localization of MMP-19 and laminin 5 at the invading tumor front but not in suprabasal epidermis of the tumor. Examination of laminin 5 processing revealed increased processing of the gamma2 chain in the medium and matrix of HaCaT-WT cells and degradation by recombinant human MMP-19 to 105-kDa and 80-kDa fragments. Parental HaCaT grown on the matrix of HaCaT-WT and HaCaT-EA cells displayed differential tyrosine phosphorylation. Using integrin blocking and stimulating antibodies we could attribute these differences to a shift from beta4-integrin-dependent signaling on the HaCaT-EA matrix toward alpha3-integrin-dependent signaling on the HaCaT-WT matrix. As a consequence, parental HaCaT showed increased migration on the matrix of HaCaT-WT cells. These data suggest that the MMP-19-dependent processing of the gamma2 chains leads to the integrin switch favoring epithelial migration and that MMP-19 actively participates in the early stages of SCC invasion.

Most of the matrix metalloproteinases (MMP) are not expressed in normal intact skin but they are upregulated in inflamed or diseased skin. The recently cloned MMP-19 is one of the few MMP members that are also expressed in healthy epidermis. In this study, we found that MMP-19 is generally coexpressed with cytokeratin 14 that is confined to keratinocytes of the stratum basale. MMP-19 was also detected in hair follicles, sebaceous glands, and eccrine sweat glands. Its expression, however, changed in cutaneous diseases exhibiting increased alternations of epidermal proliferation, such as psoriasis, eczema, and tinea. In the affected area, MMP-19 was also found in suprabasal and spinous epidermal layers. We also studied the regulation of MMP-19 expression at the protein level, as well as by using a promoter assay. The constitutive expression of MMP-19 was upregulated with phorbol myristate acetate and downregulated with retinoic acid and dexamethasone. Tumor necrosis factor-alpha, interleukin (IL)-6, TGF-beta, IL-15, IL-8, and RANTES as well as the bacterial compounds lipopolysaccharide and lipoteichoic acid did not show any profound effect in HaCaT cells. In contrast, type IV and type I collagens upregulated MMP-19 significantly. The dysregulation of MMP-19 expression in epidermis suggests its possible involvement in the perpetuation of cutaneous infections and proliferative disorders such as psoriasis.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel β-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.