Der Verlust des Signaltransduktionsmoduls des Proteins p62 führt zu einem adipösen Phänotyp bei Mäusen

Das Protein p62 ist sowohl an der Signaltransduktion, als auch an der Proteindegradation beteiligt. In dieser Arbeit wird eine Mauslinie charakterisiert, die ein p62-Protein exprimiert, dem die Aminosäuren 69 bis 251 und damit das Signaltransduktionsmodul fehlen. Durch die Entkopplung von Signaltransduktion und Proteindegradation kommt es zu einer Anreicherung freier Sauerstoffradikale und einer gesteigerten Autophagozytose in primären Zellen. Diese beiden Phänomene sind sowohl als Folge, aber auch als Ursache von Adipositas beschrieben. Mäuse die diesen p62-Gendefekt tragen entwickeln einen altersbedingten adipösen Phänotyp und können als Modellorganismus zur Untersuchungen des Metabolischen Syndroms dienen. Darüber hinaus können Studien externer Faktoren Aufschluss über die Ursache von Folgeerkrankungen von Adipositas (z.B. Herzkreislauferkrankungen und Diabetes) geben

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases

<p>Abstract</p> <p>Background</p> <p>The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex.</p> <p>Methods</p> <p>By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue.</p> <p>Results</p> <p>We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases.</p> <p>Conclusions</p> <p>The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.</p

Expression of the Major Porin Gene mspA Is Regulated in Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled σ(A) promoters of M. smegmatis. However, a 500-bp upstream fragment including P(mspA) in a transcriptional fusion with lacZ yielded only low β-galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of P(mspA) by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal mspA transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal mspA transcripts with three other 5′ untranslated region (UTRs) were three- to sixfold reduced, indicating a stabilizing role of the native 5′ UTR of mspA. Northern blot experiments revealed that the amount of mspA mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42°C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that M. smegmatis regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes

Cell surface-bound TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival pathways.

BACKGROUND: The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1-4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells. METHODOLOGY/PRINCIPAL FINDINGS: Lentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction. CONCLUSION: The results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways

Dose-dependent effect of TIMP3 on apoptosis.

<p><b>A–C:</b> Determination of the apoptosis response in different cell clones (clone 1 to 4) by caspase-3/7 activity and corresponding TIMP3 expression in these cell clones. <b>D,E:</b> Activation of initiator caspases-8 and -9 in transduced Cal78 cells with LacZ or TIMP3 cultured for 72 h. <b>F:</b> The effect of exogeneous TIMP3 on apoptosis after stimulation of Cal78 cells with recombinant human (rh) TIMP3 up to 200 nM for 96 h. Values less than p<0.05 (*) were considered statistically significant.</p

TIMP3-induced apoptosis is influenced by serum factors.

<p><b>A:</b> Influence of TIMP3 on activation of cRaf, ERK1/2, RSK1 and Akt was determined in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 by spot array measurements. <b>B:</b> Phosphorylation of cRaf, ERK1/2, RSK1 and Akt was confirmed by western blotting. <b>C:</b> Influence of serum withdrawal on apoptosis rates after 24 hours. Apoptosis in lentiviral transduced cells was measured relative to Cal78 cells cultured with 10% FCS. Values less than p<0.05 (*or °) were considered statistically significant. <b>D:</b> Influence of specific growth factors on apoptosis rates under serum-free conditions. Prior to the assessment of apoptosis, Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 were stimulated with 100 ng/ml EGF, TGF-ß or FGF-2 and cultured for 24 hours. Apoptosis was defined by caspase 3 and 7 activities. *Indicates statistical significance (p<0.05). <b>E:</b> Influence of serum withdrawal on autophagocytosis in Cal78 cells after 24 hours. Autophagocytosis in TIMP3 overexpressing CAL78 cells was determined relative to Cal78 cells transduced with a control construct (LacZ) cultured with 10% FCS, 1% FCS or ITS by Western blot analysis of the typical marker proteins LC3 and Beclin.</p

Effect of TIMP3 on apoptosis.

<p><b>A:</b> Death receptor dependent apoptosis was analyzed in Cal78 cells and Cal78 cells transduced with LacZ or TIMP3 cultured for 24 h and stimulated with 100 ng/ml FasL, TNF-a or TRAIL for 16 hours by measurement of caspase 3 and 7 activities and <b>B,C:</b> cleavage of caspase 3 and PARP in western blot analyses. GAPDH serves as an internal control. <b>D:</b> TIMP3-induced apoptosis was evaluated in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 cultured for 24 to 72 h. Apoptosis was assessed by measurement of caspase 3 and 7 activities and <b>E:</b> by histone fragmentation assay.</p

Proteoglycan-bound TIMP3 induces apoptosis.

<p><b>A:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were cultured and subsequently treated with heparinase I and III for 2 hours. Supernatants from non-treated and treated cells were transferred onto Cal78 cells for 72 hours prior assessment of apoptosis <b>B:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were incubated with or without heparinase I and III for 72 hours until evaluation of apoptotic cell death. Inactive heparinases (without CaCl₂) were used as a treatment control. Apoptosis was assessed by measurement of caspase 3 and 7 activities. Values less than p<0.05 (*) were considered statistically significant. <b>C:</b> Evaluation of TIMP3 release from cell surface of transduced cells by heparinase. Western Blot analysis of TIMP3-V5 from cell extracts and corresponding supernatants 4 hours after treatment with heparinase. <b>D:</b> Quantification of the Western Blot bands of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070709#pone-0070709-g003" target="_blank">figure 3C</a>. The band of soluble TIMP3-V5 from supernatants was shown versus bounded TIMP3-V5 from the cell lysates. <b>E:</b> Western blot analysis of the supernatants shown in figure C with a specific TIMP3 antibody. 75 ng/ml rh TIMP3 serves as an indication of the amount of TIMP3 in supernatants of Cal78 cells.</p