András Masszi

Integrity of cell-cell contacts is a critical regulator of TGF-β1-induced epithelial-to-myofibroblast transition: Role for β-catenin

Injury of the tubular epithelium and TGF-β1-induced conversion of epithelial cells to α-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-β1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-β1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca2+-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-β1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since β-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-β1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and β-catenin, but TGF-β1 selectively rescued β-catenin and stimulated the β-catenin-driven reporter TopFLASH. Moreover, chelation of free β-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-β1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a β-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-β1 are required for EMT.

Fate-determining mechanisms in epithelial-myofibroblast transition: major inhibitory role for Smad3.

Smad3 inhibits activation of the smooth muscle actin promoter and functions as a timer for myogenic programming in the epithelium.

β-Catenin and Smad3 regulate the activity and stability of myocardin-related transcription factor during epithelial–myofibroblast transition

Two novel mechanisms are shown by which injury of intercellular junctions via β-catenin promotes epithelial–myofibroblast transition. β-Catenin interacts with Smad3, thereby preventing the inhibitory effect of the latter on myocardin-related transcription factor (MRTF), and maintains MRTF stability by inhibiting Smad3-mediated, GSK-3β–dependent degradation of MRTF.

Rac, PAK and p38 regulate cell contact-dependent nuclear translocation of myocardin-related transcription factor

We investigated the mechanism whereby cell contact injury stimulates the α‐smooth muscle actin (SMA) promoter, a key process for epithelial–mesenchymal transition (EMT) during organ fibrosis. Contact disruption by low‐Ca2+ medium (LCM) activated Rac, PAK and p38 MAPK, and triggered the nuclear accumulation of myocardin‐related transcription factor (MRTF), an inducer of the SMA promoter. Dominant negative (DN) Rac, DN‐PAK, DN‐p38, or the p38 inhibitor SB203580 suppressed the LCM‐induced nuclear accumulation of MRTF and the activation of the SMA promoter. These studies define novel pathway(s) involving Rac, PAK, and p38 in the regulation of MRTF and the contact‐dependent induction of EMT.

Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation

BACKGROUND Transforming growth factor-beta (TGFbeta)-induced epithelial-myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of alpha-smooth muscle cell actin (alphaSMA) expression, a hallmark of myofibroblast formation, induced by TGFbeta in renal proximal tubular cells. METHODS Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The alphaSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular alphaSMA. To assess the regulation of the alphaSMA promoter, tubular cells were transiently transfected with a 785 bp alphaSMA promoter-luciferase reporter construct and vectors interfering with the Smad pathway. RESULTS Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFbeta-induced alphaSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38beta but not of p38alpha potently inhibited alphaSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFbeta effect, confirming the role of p38beta in the regulation of TGFbeta-induced alphaSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFbeta-induced alphaSMA synthesis. CONCLUSION TGFbeta-induced alphaSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial-mesenchymal transdifferentiation.

Smaddening complexity: the role of Smad3 in epithelial-myofibroblast transition.

Epithelial-mesenchymal transition (EMT) has emerged as a major mechanism in the pathogenesis of organ fibrosis. The epithelium has been proposed to be a significant source of matrix-producing fibroblasts and of myofibroblasts (MFs), a motile and contractile cell type hallmarked by the expression of α-smooth muscle actin (SMA). Importantly, tissue accumulation of MFs shows strong correlation with the severity and progression of fibrotic diseases. The pleiotropic cytokine transforming growth factor-β1 has been long known as the chief inducer of fibrosis, EMT and MF generation. Accordingly, receptor Smads (Smad2 and particularly Smad3), the direct targets of the activated transforming growth factor-β receptor have been implicated as critical mediators in fibrogenesis and EMT. However, evidence is accumulating that the role of Smad3 is complex and apparently controversial; in fact, Smad3 may differentially affect the various components of EMT, including the loss of epithelial markers (de-epithelialization), the production of extracellular matrix (fibrogenesis) and the expression of SMA (myogenic program). In this review, we revisit the role of Smad3 in epithelial-myofibroblast transition (EMyT). We first summarize the evidence supporting the thesis that Smad3 is a key mediator of EMT and MF generation; next, we present evidence supporting the antithesis that Smad3 is in fact a negative regulator of SMA expression and the activation of the myogenic program in the epithelium; finally, we propose a synthesis, which depicts Smad3 as a timekeeper and context-dependent modulator of EMyT. We suggest that EMyT is composed of an early, mesenchymal, Smad3-promoted phase and a late, myogenic, Smad3-inhibitable phase.

The use of a second reporter plasmid as an internal standard to normalize luciferase activity in transient transfection experiments may lead to a systematic error

beta-galactosidase reporter plasmids containing different viral or minimal promoters are commonly used to correct variable transfection efficiencies in transient transfection experiments. The transcriptional activity of these promoters is thought to be stable under most circumstances. To determine if expression of beta-galactosidase from the commonly used beta-galactosidase plasmids remains stable upon stimulation of the cells with agonists we performed transient transfection experiments. CHO cells stably expressing the rat AT(1A) receptor were transfected with RSVbeta- or CMVbeta- or pTKbeta plasmids alone or together with a reporter construct in which luciferase transcription is driven by the c-fos promoter. Luciferase and/or beta-galactosidase activity was measured from the lysate of cells treated with angiotensin II or serum. We found that agonists increased the transcriptional activity of the different beta-galactosidase plasmids. The effect of angiotensin II and serum was different on the different promoters. Finally, cotransfection of other plasmids also modulated beta-galactosidase activity. These agonist induced variations of beta-galactosidase activity may influence the analysis and interpretation of the results in a systematic manner. Consequently we conclude that the use of a second reporter system to control for transfection efficiency in certain types of experiments may lead to a systematic error and is questionable as a general procedure.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF translocation, since siRNA-mediated GEF-H1 downregulation suppresses these responses. While the osmotically induced RhoA activation promotes nuclear MRTF accumulation, the concomitant activation of p38 MAP kinase mitigates this effect. Moderate hyperosmotic stress (600 mosM) drives MRTF-dependent transcription through the cis-element CArG box. Silencing or pharmacological inhibition of MRTF prevents the osmotic stimulation of CArG-dependent transcription and renders the cells susceptible to osmotic shock-induced structural damage. Interestingly, strong hyperosmolarity promotes proteasomal degradation of MRTF, concomitant with apoptosis. Thus, MRTF is an osmosensitive and osmoprotective transcription factor, whose intracellular distribution is regulated by the GEF-H1/RhoA/ROK and p38 pathways. However, strong osmotic stress destabilizes MRTF, concomitant with apoptosis, implying that hyperosmotically induced cell death takes precedence over epithelial-myofibroblast transition, a potential consequence of MRTF-mediated phenotypic reprogramming.

Extracellular Signal-Regulated Kinase and the Small GTP-Binding Protein p21Rac1 Are Involved in the Regulation of Gene Transcription by Angiotensin II

To study the role of extracellular-signal-regulated kinase (ERK) cascade and the small GTP-ase proteins in the activation of the c-fos promoter by angiotensin II (AII), transient transfection experiments were performed in CHO cells stably expressing the rat AT1A receptor. In this system AII activated ERK in 1 min and also increased the transcriptional activity of the c-fos promoter-luciferase reporter gene construct. The activation of the promoter proved to be dependent on the Ras-Raf-ERK cascade as cotransfection of expression vectors known to specifically inhibit this cascade blocked the effect of AII. Dominant-negative p21Rac1 mutant partially blocked the activation of the c-fos promoter by AII. However, activation of the c-fos promoter was independent of protein kinase C (PKC) as bisindolylmaleimide I, a specific PKC inhibitor did not block the effect of AII. These results suggest that AII activates the transcription of the c-fos through the Ras-Raf-ERK cascade. Furthermore, p21Rac1 is involved in the modulation of the c-fos promoter by AII.

Thalidomidkezelés relabált, diffúz nagy B-sejtes lymphomában idős betegekben. Három eset bemutatása

Absztrakt: A diffuz nagy B-sejtes lymphoma (DLBCL) a leggyakoribb malignus lymphomatipus, amely a non-Hodgkin-lymphomak (NHL) 31%-at teszi ki. A standard kemoterapias protokollok jelentős toxicitasuk miatt nem vagy nehezen alkalmazhatoak az idősebb betegpopulacioban, kulonosen a relabalt esetekben, ahol kurativ lehetőseget leginkabb a nagy dozisu kezeles es a ver-őssejtatultetes jelenthetne. Egyre tobb adat lat napvilagot a thalidomid es az ujabb IMiD-ek (lenalidomid, pomalidomid) kedvező hatasairol az NHL kezeleseben, amelyek reszei a standard kezelesnek myeloma multiplexben es myelodysplasias szindromaban. Az IMiD-ek kiterjedt anti-angiogenetikus, immunmodulans es gyulladasellenes hatasuknak koszonhetik az egyre szeleskorűbb alkalmazast. 2010-ben azonositottak az E3-ubiquitin-ligaz-komplex egyik komponenset, a cereblont, amely a thalidomid molekularis tamadasi pontja. Az irodalomban kozolt eredmenyek alapjan thalidomidkezelest inditottunk harom relabalt, idős DLBCL-es betegunknel. Ket esetben kozponti i...