Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

[How did the survival of acute myeloid leukemia change over the last ten years in our unit?] / Hogyan változott az akut myeloid leukaemiás betegek túlélése a terápiás lehetoségek bovülésével az elmúlt 10 évben klinikánkon?

INTRODUCTION: Acute myeloid leukemia (AML) is a hematological malignancy with high mortality rate. The treatment is especially challenging in patients older than 65 years, which is the large majority of those. For patients unfit for intensive chemotherapy regimens, only palliative cytoreduction and basic supportive care used to be the options in our unit. However, from 2018, the azacitidine-venetoclax combination has been a new therapeutic alternative. This treatment resulted in marked survival benefit in clinical trials, however, its impact on the daily clinical practice and the entire patient population is unclear. OBJECTIVE: Our goal was to evaluate how the application of azacitidine-venetoclax changed the treatment and survival of AML patients in our practice. METHOD: We retrospectively analyzed the available clinical data of all AML patients treated consecutively between January 1, 2011 and December 31, 2021 at the 3rd Department of Internal Medicine (from 2020 onward called Department of Internal Medicine and Hematology), examining their treatment depending on the time period of therapy (2011-2017 and 2018-2021). Patients with acute promyelocytic leukemia were excluded. RESULTS: 423 patients were diagnosed during this period. The number of cases showed a marked increase: in the first 7 years of our study, 184 patients were diagnosed, while this rose to 239 during the subsequent 4 years. The median age of patients was 67.6 years, with more than 60% of patients aged over 65. An improving trend can be observed in the overall survival: between 2011 and 2017, the median overall survival was 4.8 ± 0.9 months, while between 2018 and 2021, it was 8.3 ± 1.4 months (p = 0.051). Moreover, in the case of patients over 65 there was a significant overall survival improvement: 3.1 ± 0.5 vs. 4.9 ± 0.6 months (p = 0,01). The main factor behind this improvement could be that a large proportion of over 65 patients previously only fit for supportive care could now be treated with azacitidine-venetoclax: the percentage of actively treated patients grew from 57.1% to 75.3% in the second period. CONCLUSION: The survival of patients unfit for curative therapy and older than 65 showed a steady increase which can be attributed to the introduction of new therapeutic alternatives. Orv Hetil. 2023; 164(45): 1787-1794.

[Fertility preservation in female cancer patients.] / Daganatos nobetegek termékenységének megorzése.

In Hungary, an average of 2066 women under the age of 40 are diagnosed with cancer each year according to data from the National Cancer Registry. Approximately two-thirds of these patients require gonadotoxic treatment for their disease, which could potentially reduce their chances of future conception and childbirth. Currently, there are no professional guidelines on fertility preservation in Hungary, however, it is important to inform patients about their options. In our previous paper, we presented the gonadotoxic effects of oncotherapies and the currently available fertility preservation techniques. This second paper provides current treatment methods and recommends fertility preservation techniques in different cancer types. The success of an oncofertility program relies heavily on the effective communication and collaboration between oncologists and reproductive specialists involved in fertility preservation. This paper may be the first step in elaborating a guideline towards improving access to oncofertility services and ultimately improving the quality of life for young cancer survivors in Hungary. Orv Hetil. 2023; 164(29): 1134-1145.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax.

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Revealing a Phenotypical Appearance of Ibrutinib Resistance in Patients With Chronic Lymphocytic Leukaemia by Flow Cytometry.

Background: Ibrutinib is widely known as an effective and well-tolerated therapeutical choice of the chronic lymphocytic leukaemia (CLL). However, acquired resistance may occur during the treatment, causing relapse. Early detection of ibrutinib resistance is an important issue, therefore we aimed to find phenotypic markers on CLL cells the expression of which may correlate with the appearance of ibrutinib resistance. Methods: We examined 28 patients' peripheral blood (PB) samples (treatment naïve, ibrutinib sensitive, clinically ibrutinib resistant). The surface markers' expression (CD27, CD69, CD86, CD184, CD185) were measured by flow cytometry. Furthermore, the BTKC481S resistance mutation was assessed by digital droplet PCR. Moreover, the CLL cells' phenotype of a patient with acquired ibrutinib resistance was observed during the ibrutinib treatment. Results: The expression of CD27 (p = 0.030) and CD86 (p = 0.031) became higher in the clinically resistant cohort than in the ibrutinib sensitive cohort. Besides, we found that high CD86 and CD27 expressions were accompanied by BTKC481S mutation. Our prospective study showed that the increase of the expression of CD27, CD69 and CD86 was noticed ahead of the clinical resistance with 3 months. Conclusion: Our study suggests that the changes of the expression of these markers could indicate ibrutinib resistance and the examination of these phenotypic changes may become a part of the patients' follow-up in the future.

Quantitative Analysis and Monitoring of EZH2 Mutations Using Liquid Biopsy in Follicular Lymphoma.

Recent advances in molecular technologies enable sensitive and quantitative assessment of circulating tumor DNA, offering a noninvasive disease monitoring tool for patients with malignant disorders. Here, we demonstrated on four follicular lymphoma cases that circulating tumor DNA based EZH2 mutation analysis performed by a highly sensitive droplet digital PCR method may be a valuable treatment monitoring approach in EZH2 mutant follicular lymphoma. EZH2 variant allele frequencies changed in parallel with the volume of metabolically active tumor sites observed on 18F-fluorodeoxyglucose positron emission tomography combined with computer tomography (PET-CT) scans. Variant allele frequencies of EZH2 mutations decreased or were eliminated rapidly upon successful treatment, with treatment failure being associated with elevated EZH2 variant allele frequencies. We also demonstrated spatial heterogeneity in a patient with two different EZH2 mutations in distinct anatomical sites, with both mutations simultaneously detected in the liquid biopsy specimen. In summary, circulating tumor DNA based EZH2 mutation analysis offers a rapid, real-time, radiation-free monitoring tool for sensitive detection of EZH2 mutations deriving from different anatomical sites in follicular lymphoma patients receiving immunochemotherapy.

MASP-1 Increases Endothelial Permeability.

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs-for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development.

[Thalidomide therapy in relapsed diffuse large B-cell lymphoma in elderly patients. Three cases]. / Thalidomidkezelés relabált, diffúz nagy B-sejtes lymphomában idos betegekben. Három eset bemutatása.

Diffuse large B-cell lymphoma (DLBCL), a high-grade lymphoproliferative disease, is the most common lymphoma in adults, representing 31% of non-Hodgkin lymphomas (NHL). In elderly patients treatment is problematic because of the high toxicity of standard chemotherapy protocols, especially in relapsed cases, where high-dose chemotherapy and haematopoietic stem cell transplantation would be the best choice. More and more data is becoming available on alternative treatment of refractory/relapsed NHL, including studies on the positive effect of thalidomide and second generation IMiDs in DLBCL, which are already part of the standard treatment protocol in myeloma multiplex and myelodysplasia. The broadening use of IMiDs is due to their anti-angiogenetic, immunmodulatory and anti-inflammatory properties. In addition, a component of the E3-ubiquitin ligase complex, named cereblon, has been described in 2010 as the molecular effector of the thalidomide signal transduction pathway. We initiated thalidomide treatment in three elderly patients with relapsed DLBCL. In two cases, patients had CNS involvement, in the third case the patient had primary mediastinal disease. Patients received 100 mg thalidomide in combination with corticosteroids. Two patients showed an excellent response reaching complete remission on imaging; these patients are progression-free 12 and 20 months after the beginning of treatment. One patient with CNS involvement progressed and deceased despite therapy. According to the literature, IMiDs have significant activity in relapsed DLBCL. Our case-report presents promising results in an elderly patient population with aggressive relapsed NHL that usually has very poor outcome, as high-toxicity treatment cannot be given to these patients. Consequently, because of its efficiency, low-cost and low-toxicity, it is recommended to consider thalidomide therapy in elderly patients with high-grade DLBCL. Orv Hetil. 2017; 158(41): 1642-1648.

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.

ß-catenin and Smad3 regulate the activity and stability of myocardin-related transcription factor during epithelial-myofibroblast transition.

Injury to the adherens junctions (AJs) synergizes with transforming growth factor-ß1 (TGFß) to activate a myogenic program (α-smooth muscle actin [SMA] expression) in the epithelium during epithelial-myofibroblast transition (EMyT). Although this synergy plays a key role in organ fibrosis, the underlying mechanisms have not been fully defined. Because we recently showed that Smad3 inhibits myocardin-related transcription factor (MRTF), the driver of the SMA promoter and many other CC(A/T)-rich GG element (CArG) box-dependent cytoskeletal genes, we asked whether AJ components might affect SMA expression through interfering with Smad3. We demonstrate that E-cadherin down-regulation potentiates, whereas ß-catenin knockdown inhibits, SMA expression. Contact injury and TGFß enhance the binding of ß-catenin to Smad3, and this interaction facilitates MRTF signaling by two novel mechanisms. First, it inhibits the Smad3/MRTF association and thereby allows the binding of MRTF to its myogenic partner, serum response factor (SRF). Accordingly, ß-catenin down-regulation disrupts the SRF/MRTF complex. Second, ß-catenin maintains the stability of MRTF by suppressing the Smad3-mediated recruitment of glycogen synthase kinase-3ß to MRTF, an event that otherwise leads to MRTF ubiquitination and degradation and the consequent loss of SRF/MRTF-dependent proteins. Thus ß-catenin controls MRTF-dependent transcription and emerges as a critical regulator of an array of cytoskeletal genes, the "CArGome."

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-ß(1) 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-ß 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.

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

Epithelial-myofibroblast (MF) transition (EMyT) is a critical process in organ fibrosis, leading to alpha-smooth muscle actin (SMA) expression in the epithelium. The mechanism underlying the activation of this myogenic program is unknown. We have shown previously that both injury to intercellular contacts and transforming growth factor beta (TGF-beta) are indispensable for SMA expression (two-hit model) and that contact disruption induces nuclear translocation of myocardin-related transcription factor (MRTF). Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-beta-responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3. In this study, we show that the synergy between injury and TGF-beta exclusively requires CArG elements. Surprisingly, Smad3 inhibits MRTF-driven activation of the SMA promoter, and Smad3 silencing renders injury sufficient to induce SMA expression. Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program. Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

Transforming growth factor-beta-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38beta 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.

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 alpha-smooth muscle actin (SMA) promoter, a key process for epithelial-mesenchymal transition (EMT) during organ fibrosis. Contact disruption by low-Ca(2+) 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.

Cell contact-dependent regulation of epithelial-myofibroblast transition via the rho-rho kinase-phospho-myosin pathway.

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell-cell junctions is a prerequisite for transforming growth factor-beta1 (TGF-beta1)-induced transdifferentiation of kidney tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca(2+) removal-induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-beta1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-beta1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca(2+)-removal- +/- TGF-beta1-induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC-dependent nuclear accumulation of MRTF.

Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin.

Injury of the tubular epithelium and TGF-beta1-induced conversion of epithelial cells to alpha-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-beta 1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-beta 1 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 Ca(2+)-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-beta 1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since beta-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-beta 1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and beta-catenin, but TGF-beta1 selectively rescued beta-catenin and stimulated the beta-catenin-driven reporter TopFLASH. Moreover, chelation of free beta-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-beta1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a beta-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-beta 1 are required for EMT.

Angiotensin II activates the human renin promoter in an in vitro model: the role of c-Jun-N-terminal kinase.

BACKGROUND: Angiotensin II (Ang II) down-regulates renin expression in juxtaglomerular cells, however, recent experimental evidence obtained in transgenic mice suggested that Ang II may "paradoxically" increase transcriptional activity of the proximal renin promoter. METHODS: To dissect signalling mechanisms contributing to the up-regulation of the proximal renin promoter by Ang II, porcine proximal tubular cells stably expressing the rabbit AT(1) receptor (LLC-PK/AT(1)) were transiently transfected with a luciferase reporter construct containing the 582 bp long piece of the human renin promoter. Activation of mitogen-activated protein kinases (MAPK) was detected by western blotting using phospho-MAPK-specific antibodies. RESULTS: Ang II dose-dependently stimulated the transcriptional activity of the human renin promoter (10(-7) M Ang II: 3.50+/-1.25-fold stimulation). In these cells Ang II activated both extracellular signal-regulated kinase (ERK) and the c-Jun-N-terminal kinase (JNK). Inhibition of the ERK cascade did not reduce the stimulation of the renin promoter by Ang II, however, two expression vectors designed to inhibit the JNK pathway, the dominant negative JNK and the Jun-kinase interacting peptide inhibited the fold stimulation induced by Ang II (2.75+/-0.69 vs 1.6+/-0.23 and 1.8+/-0.34, respectively; P<0.01). Furthermore, genistein, a tyrosine kinase inhibitor, blocked the effect of Ang II both on the JNK and the promoter activation. CONCLUSION: Ang II may have a stimulatory effect on the proximal renin promoter in proximal tubular cells and this effect is mediated by tyrosine kinases and the JNK cascade.

[Role of mitogen-activated protein kinase cascade in the intracellular signaling of angiotensin II]. / A mitogén által aktivált proteinkináz kaszkádok szerepe az angiotenzin II intracelluláris jelátvitelében.

It has been recently recognized that angiotensin II (Ang II), in addition to its role in the regulation of the salt-fluid homeostasis is also a growth factor and contributes to the regulation of cellular hypertrophy or proliferation and extracellular matrix (ECM) formation. This article gives an overview on the role of mitogen activated protein kinase (MAPK) cascades in transducing the growth factor-like effects of Ang II.