Baharia Mograbi

Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane Is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Activity

Macroautophagy (hereafter referred to as autophagy) has emerged as a key tumor suppressor pathway. During this process, the cytosolic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes to become autolysosomes where their contents are finally degraded. Although a reduced autophagy has been shown in human tumors or in response to oncogenes and carcinogens, the underlying mechanism(s) remain(s) unknown. Here, we show that widely used carcinogen Lindane promotes vacuolation of Sertoli cells. By electron and immunofluorescent microscopy analyses, we showed that these structures are acid autolysosomes, containing cellular debris, and labeled by LC3, Rab7, and LAMP1, markers of autophagosomes, late endosomes, and lysosomes, respectively. Such Lindane-induced vacuolation results from significant delay in autophagy degradation, in relation with a decline of the lysosomal activity of aryl sulfatase A. At molecular level, we show that this defect in autolysosomal maturation is independent of mammalian target of rapamycin and p38 inhibitions. Rather, the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is required for Lindane to disrupt the autophagic pathway. Most importantly, we provide the first evidence that sustained activation of ERK pathway is sufficient to commit cell to autophagic vacuolation. Taken together, these findings strongly support that the aberrant sustained activation of ERK by the carcinogen Lindane disrupts the maturation of autophagosomes into functional autolysosomes. Our findings therefore suggest the possibility that high constitutive ERK activity found in all cancers may provide a malignant advantage by impeding the tumor suppressive function of autophagy.

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease

Susceptibility to Crohns disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohns disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohns disease–associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohns disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.

Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Escherichia coli-infected T84 cells.

ABSTRACT Use of the nonpathogenic yeast Saccharomyces boulardiiin the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenic Escherichia coli (EPEC)-associated disease demonstrates that S. boulardii abrogated the alterations induced by an EPEC strain on transepithelial resistance, [3H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence of S. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence of S. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence of S. boulardii. Altogether, this study demonstrated that S. boulardii exerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.

Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens.

Macroautophagy (hereafter referred to as autophagy) is the major degradative pathway of long-lived proteins and organelles that fulfils key functions in cell survival, tissue remodeling and tumor suppression. Consistently, alterations in autophagy have been involved in a growing list of pathologies including toxic injury, infections, neurodegeneration, myopathies and cancers. Although critical, the molecular mechanisms that control autophagy remain largely unknown. We have recently exploited the disruption of autophagy by environmental carcinogens as a powerful model to uncover the underlying signaling pathways. Our work published in Cancer Research revealed that the sustained activation of the MAPK ERK pathway by the carcinogen Lindane or the MEK1+ oncogene alters autophagy selectively at the maturation step resulting in the accumulation of large defective autolysosomes. Consistent with our findings, a similar defect is observed with other common xenobiotics such as dichlorodiphenyltrichloroethane and biphenol A that specifically activate ERK. Conversely, Pentachlorophenol that activates both ERK and p38, fails to induce autophagic vacuolation. In addition, evidence is provided that abrogation of p38 by SB203580 is sufficient to interfere with the normal autophagic maturation step. Altogether, these findings underscore the critical role played by MAPK ERK and p38 in the tight control of the autophagy process at the maturation step. Addendum to: Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Activity E. Corcelle, M. Nebout, S. Bekri, N. Gauthier, P. Hofman, P. Poujeol, P. Fénichel and B. Mograbi Cancer Res 2006; 66:6861-70

Cadmium-Induced Autophagy in Rat Kidney: An Early Biomarker of Subtoxic Exposure

Environmental exposures to cadmium (Cd) are a major cause of human toxicity. The kidney is the most sensitive organ; however, the natures of injuries and of adaptive responses have not been adequately investigated, particularly in response to environmental relevant Cd concentrations. In this study, rats received a daily ip injection of low CdCl₂ dose (0.3 mg Cd/kg body mass) and killed at 1, 3, and 5 days of intoxication. Functional, ultrastructural, and biochemical observations were used to evaluate Cd effects. We show that Cd at such subtoxic doses does not affect the tubular functions nor does it induce apoptosis. Meanwhile, Cd accumulates within lysosomes of proximal convoluted tubule (PCT) cells where it triggers cell proliferation and autophagy. By developing an immunohistochemical assay, a punctate staining of light chain 3-II is prominent in Cd-intoxicated kidneys, as compared with control. We provide the evidence of a direct upregulation of autophagy by Cd using a PCT cell line. Compared with the other heavy metals, Cd is the most powerful inducer of endoplasmic reticulum stress and autophagy in PCT cells, in relation to the hypersensitivity of PCT cells. Altogether, these findings suggest that kidney cortex adapts to subtoxic Cd dose by activating autophagy, a housekeeping process that ensures the degradation of damaged proteins. Given that Cd is persistent within cytosol, it might damage proteins continuously and impair at long-term autophagy efficiency. We therefore propose the autophagy pathway as a new sensitive biomarker for renal injury even after exposure to subtoxic Cd doses.

Impaired Gap Junction Connexin43 in Sertoli Cells of Patients with Secretory Azoospermia: A Marker of Undifferentiated Sertoli Cells

Gap junctions are intercellular channels formed of connexins (Cx) at appositional plasma membranes between adjacent cells that have been involved in the control of cell proliferation and differentiation. Altered Cx expression is implicated consistently in several human diseases and in tumorigenesis. Although Cx43 plays a critical role in Sertoli cell control of spermatogenesis, there is no evidence of its altered expression in human testicular pathologies. We show here that Cx43 mRNA expression was significantly reduced in testes of infertile patients with secretory azoospermia (p < 0.05) compared with testes displaying normal spermatogenesis (excretory azoospermic patients). In Sertoli cell-only syndrome, in situ hybridization and immunohistochemistry analyses indicated that Cx43 mRNA and protein were undetectable in Sertoli cells but were still present in the interstitial compartment. In a rat model of Sertoli cell-only syndrome, the lack of Cx43 in Sertoli cells was associated with an impairment of gap junction intercellular communication between adjacent Sertoli cells. These results reveal that Cx43 mRNA and protein expression are markedly impaired in Sertoli cells of infertile patients. This defect could be a new functional marker of undifferentiated Sertoli cells and could be related to the increased risk of testicular cancer recently described in the population of infertile men.

Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection.

ABSTRACT Enteropathogenic Escherichia coli (EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (ΔeaeA) and type III secretion apparatus mutant strain CVD452 (ΔescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded α‐lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double‐membrane‐enclosed vesicles typical of macroautophagy. In addition, HAMLET caused a change from uniform (LC3‐I) to granular (LC3‐II) staining in LC3‐GFP‐transfected cells reflecting LC3 translocation during macroautophagy, and this was blocked by the macroautophagy inhibitor 3‐methyladenine. HAMLET also caused accumulation of LC3‐II detected by Western blot when lysosomal degradation was inhibited suggesting that HAMLET caused an increase in autophagic flux. To determine if macroautophagy contributed to cell death, we used RNA interference against Beclin‐1 and Atg5. Suppression of Beclin‐1 and Atg5 improved the survival of HAMLET‐treated tumor cells and inhibited the increase in granular LC3‐GFP staining. The results show that HAMLET triggers macroautophagy in tumor cells and suggest that macroautophagy contributes to HAMLET‐induced tumor cell death.

MiR-129-5p is required for histone deacetylase inhibitor-induced cell death in thyroid cancer cells.

The molecular mechanism responsible for the antitumor activity of histone deacetylase inhibitors (HDACi) remains elusive. As HDACi have been described to alter miRNA expression, the aim of this study was to characterize HDACi-induced miRNAs and to determine their functional importance in the induction of cell death alone or in combination with other cancer drugs. Two HDACi, trichostatin A and vorinostat, induced miR-129-5p overexpression, histone acetylation and cell death in BCPAP, TPC-1, 8505C, and CAL62 cell lines and in primary cultures of papillary thyroid cancer (PTC) cells. In addition, miR-129-5p alone was sufficient to induce cell death and knockdown experiments showed that expression of this miRNA was required for HDACi-induced cell death. Moreover, miR-129-5p accentuated the anti-proliferative effects of other cancer drugs such as etoposide or human α-lactalbumin made lethal for tumor cells (HAMLET). Taken together, our data show that miR-129-5p is involved in the antitumor activity of HDACi and highlight a miRNA-driven cell death mechanism.

Disrupted traffic of connexin 43 in human testicular seminoma cells: overexpression of Cx43 induces membrane location and cell proliferation decrease.

Connexins, the constitutive proteins of gap junctions, are considered to be tumour suppressive agents and are often impaired in the tumourigenic processes. In the present study, the expression of connexin 43 (Cx43), which is involved in the control of spermatogenesis through Sertoli/germ cell coupling, has been investigated in human testicular seminoma cells (tumours and the JKT‐1 cell line). Cx43 was immunolocalized in the Golgi apparatus without membrane expression and was detected by immunoblotting in JKT‐1 as exclusive 70 kD bands. No mutation could be found by sequencing the transcript obtained by RT‐PCR. Transfection with a Cx43‐V5 vector reproduced the same gel shift, identifying these 70 kD bands as Cx43. The Cx43‐70 kD bands were also expressed in normal testicular tissue, associated with the classical 43 kD isoforms. Stable transfection of JKT‐1 with a Cx43‐GFP vector allowed restoration of Cx43 membrane expression, functional cell coupling, and inhibition of the cell proliferation rate. Storage of Cx43 in the Golgi apparatus may correspond during spermatogenesis to an intermittent physiological process that becomes permanent in malignant seminoma cells as a result of the tumourigenic process. By preventing Cx43 membrane expression, this disrupted traffic may itself participate in tumour promotion. Copyright