Autophagy activation is required for influenza A virus-induced apoptosis and replication.

Autophagy and apoptosis are two major interconnected host cell responses to viral infection, including influenza A virus (IAV). Thus, delineating these events could facilitate the development of better treatment options and provide an effective anti-viral strategy for controlling IAV infection. We used A549 cells and mouse embryonic fibroblasts (MEF) to study the role of virus-induced autophagy and apoptosis, the cross-talk between both pathways, and their relation to IAV infection [ATCC strain A/Puerto Rico/8/34(H1N1) (hereafter; PR8)]. PR8-infected and mock-infected cells were analyzed by immunoblotting, immunofluorescence confocal microscopy, electron microscopy and flow cytometry (FACS). We found that PR8 infection simultaneously induced autophagy and apoptosis in A549 cells. Autophagy was associated with Bax and Bak activation, intrinsic caspase cleavage and subsequent PARP-1 and BID cleavage. Both Bax knockout (KO) and Bax/Bak double knockout MEFs displayed inhibition of virus-induced cytopathology and cell death and diminished virus-mediated caspase activation, suggesting that virus-induced apoptosis is Bax/Bak-dependent. Biochemical inhibition of autophagy induction with 3-methyladenine blocked both virus replication and apoptosis pathways. These effects were replicated using autophagy-refractory Atg3 KO and Atg5 KO cells. Taken together, our data indicate that PR8 infection simultaneously induces autophagy and Bax/caspase-dependent apoptosis, with autophagy playing a role to support PR8 replication, in part, by modulating virus-induced apoptosis.

An additive interaction between the NFkappaB and estrogen receptor signalling pathways in human endometrial epithelial cells.

BACKGROUND: Human embryo implantation is regulated by estradiol (E2), progesterone and locally produced mediators including interleukin-1beta (IL-1beta). Interactions between the estrogen receptor (ER) and NF kappa B (NFkappaB) signalling pathways have been reported in other systems but have not been detailed in human endometrium. METHODS AND RESULTS: Real-time PCR showed that mRNA for the p65 and p105 NFkappaB subunits is maximally expressed in endometrium from the putative implantation window. Both subunits are localized in the endometrial epithelium throughout the menstrual cycle. Reporter assays for estrogen response element (ERE) activity were used to examine functional interactions between ER and NFkappaB in telomerase immortalized endometrial epithelial cells (TERT-EEC). E2 and IL-1beta treatment of TERT-EECs enhances ERE activity by a NFkappaB and ER dependent mechanism; this effect could be mediated by ERalpha or ERbeta. E2 and IL-1beta also positively interact to increase endogenous gene expression of prostaglandin E synthase and c-myc. This is a gene-dependent action as there is no additive effect on cyclin D1 or progesterone receptor expression. CONCLUSION: In summary, we have established that NFkappaB signalling proteins are expressed in normal endometrium and report that IL-1beta can enhance the actions of E2 in a cell line derived from healthy endometrium. This mechanism may allow IL-1beta, possibly from the developing embryo, to modulate the function of the endometrial epithelium to promote successful implantation, for example by regulating prostaglandin production. Aberrations in the interaction between the ER and NFkappaB signalling pathways may have a negative impact on implantation contributing to pathologies such as early pregnancy loss and infertility.

AhR-agonist-induced transcriptional changes of genes involved in thyroid function in primary porcine thyrocytes.

The Ah receptor (AhR) is a ligand transcription factor mediating toxic effects of chemicals such as dioxins. The 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar polychlorinated biphenyl 126 (PCB 126) are member of the polyhalogenated aromatic hydrocarbons family exerting a variety of toxic effects in a tissue-specific and species-specific manner including thyroid function. In the present study, we aimed to investigate the effects of TCDD (1 and 10 nM) and dioxin-like PCB 126 (306 nM) on the AhR signaling pathway and on the gene expression profiles of key factors involved in thyroid function, including thyroglobulin (TG), thyroid peroxidase (TPO), the sodium iodide symporter (NIS), TSH receptor (TSHR), and cathepsins (Cat B and L), using a primary porcine thyrocyte culture as the experimental model. AhR and ARNT expression was detected both as mRNA and on the protein level. Expression did not vary upon treatment with either TCDD or PCB 126. However, treatment with TCDD and PCB 126 induced an AhR signaling response, as indicated by the expression of the AhR-target gene cytochrome P-450 1A1 (CYP1A1). Both 10 nM TCDD and PCB 126 treatment induced a significant downregulation in the expression of NIS and cathepsin B without affecting any of the other parameters investigated. In conclusion, these data indicate that (a) thyrocytes are targets of TCDD and TCDD-like compounds and (b) there is evidence for two independent most likely AhR-mediated molecular mechanisms, by which these compounds negatively interfere with thyroid function.

Comparing a thyroid prognostic nomogram to the existing staging systems for prediction risk of death from thyroid cancers.

OBJECTIVE: Thyroid prognostic nomogram can be applied across different histological types for predicting the individualized risk of death from thyroid cancer. The objective of this study was to compare the strength of our recently published thyroid prognostic nomogram with 12 existing staging systems to predict the risk of death from thyroid cancer. METHOD: This study included 1900 thyroid cancer patients, from a population based cohort of 2296 patients, on whom adequate staging information was available. Competing risk sub-hazard models were used to compare 12 pre-existing prognostic models with the nomogram model. Their relative strengths for prediction of patients' individualized risks of death from thyroid cancer were compared using Akaike information criterion (AIC), delta AIC, and concordance index. R version 3.2.2 was used to analyze the data. RESULTS: Our cohort of 450 males and 1450 females included 1796 (93.4%) differentiated thyroid cancers. Amongst the compared models, thyroid prognostic nomogram model appeared to be better than other models for predicting the risk of death from all non-anaplastic thyroid cancer (concordance index = 94.4), differentiated thyroid cancer (concordance index = 94.1) and papillary thyroid cancer (concordance index = 94.7). The difference from next best staging systems was most pronounced in non-anaplastic thyroid cancer (delta AIC = 114.8), followed by differentiated thyroid cancer (delta AIC = 35.6) and papillary thyroid cancer (delta AIC = 8.4). CONCLUSIONS: Thyroid prognostic nomogram model was found to be better than the other models compared for predicting risk of death from thyroid cancer.

Molecular actions of polyhalogenated arylhydrocarbons (PAHs) in female reproduction.

Polyhalogenated aromatic arylhydrocarbons (PAHs) such as polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), the polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent lipophilic pollutants, which affect female fertility resulting in severe reproductive dysregulation, including anovulation, reduced conception rates, abortion, menstrual abnormalities and developmental defects of female reproductive tissues. Many PAHs exert their effects by activating a family of basic helix-loop-helix (bHLH) transcription factors, the arylhydrocarbon receptor (AhR) and the arylhydrocarbon receptor nuclear translocator (ARNT), which result in the expression of AhR target molecules. Complex interactions between PAH-mediated AhR activation and ER signalling pathways have been discovered which may contribute to the developmental malformations, impact on reproductive dysfunctions and promote carcinogenic dedifferentiation of tissues within the female reproductive tract. This review will focus on the multifaceted roles of PAHs in key organs of the female reproductive tract, the ovary, uterus/ endometrium and the mammary gland. The complexity and diversity of actions unleashed by PAHs in these female reproductive tissues identify these environmental pollutants as important endocrine disrupting toxicants impacting on female fertility.

Regulation of functional steroid receptors and ligand-induced responses in telomerase-immortalized human endometrial epithelial cells.

Information on the regulation of steroid hormone receptors and their distinct functions within the human endometrial epithelium is largely unavailable. We have immortalized human primary endometrial epithelial cells (EECs) isolated from a normal proliferative phase endometrium by stably transfecting the catalytic subunit (hTERT) of the human telomerase complex and cultured these hTERT-EECs now for over 350 population doublings. Active hTERT was detected in hTERT-EECs employing the telomerase repeat amplification assay protocol. hTERT-EECs revealed a polarized, non-invasive epithelial phenotype with apical microvilli and production of a basal lamina when grown on a three-dimensional collagen-fibroblast lattice. Employing atomic force microscopy, living hTERT-EECs were shown to produce extracellular matrix (ECM) components and ECM secretion was modified by estrogen and progesterone (P4). hTERT-EECs expressed inducible and functional endogenous estrogen receptor-alpha (ER-alpha) as demonstrated by estrogen response element reporter assays and induction of P4 receptor (PR). P4 treatment down-regulated PR expression, induced MUC-1 gene activity and resulted in increased ER-beta transcriptional activity. Gene activities of cytokines and their receptors interleukin (IL)-6, leukemia inhibitory factor (LIF), IL-11 and IL-6 receptor (IL6-R), LIF receptor and gp130 relevant to implantation revealed a 17 beta-estradiol (E2)-mediated up-regulation of IL-6 and an E2- and P4-mediated up-regulation of IL6-R in hTERT-EECs. Thus, hTERT-EECs may be regarded as a novel in vitro model to investigate the role of human EECs in steroid hormone-dependent normal physiology and pathologies, including implantation failure, endometriosis and endometrial cancer.

Human medullary thyroid carcinoma: a source and potential target for relaxin-like hormones.

We investigated the expression of H1, H2 relaxin and INSL-3, mRNA and protein, and LGR7 and LGR8 transcripts in human C-cell hyperplasia, primary medullary thyroid carcinoma (MTC) tissues, MTC metastases, and the human MTC-TT and mouse MTC-M cell lines. Relaxin-like peptide hormones were detected in C-cell hyperplasia and in MTC tissues, but were absent in human normal parafollicular C-cells of benign goiter tissues. In contrast to calcitonin, mRNA, and immunoreactive protein, no differences in the expression of relaxin and INSL3 were observed in MTC tissues of different pTNM classification or between primary and metastatic MTC tissues studied. All MTC tissues constitutively expressed LGR7 and LGR8 transcripts. Thus, relaxin-like hormones appear to be present early during C-cell hyperplasia and potentially functional relaxin/INSL3 ligand-receptor systems are present in human MTC tissues and in MTC cell lines.

Autophagy is a regulator of TGF-ß1-induced fibrogenesis in primary human atrial myofibroblasts.

Transforming growth factor-ß(1) (TGF-ß(1)) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-ß(1) may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-ß(1)-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-ß(1) to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-ß(1) promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-ß(1) in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3ß indicated the localization of punctate LC3ß with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-ß(1)-induced autophagy is required for the fibrogenic response in hATMyofbs.

Dual specificity antibodies using a double-stranded oligonucleotide bridge.

The covalent conjugation of oligonucleotides to antibody Fab' fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.

Placental localization of relaxin in the pregnant mare.

In situ hybridization employing a cRNA probe derived from a 428-bp fragment of equine relaxin was used to localize relaxin mRNA, and immunocytochemistry was used to localize relaxin itself, in tissues of the placenta-endometrium interface recovered between 33 and 153 days of gestation from mares carrying intraspecific horse, interspecific mule and extraspecific donkey conceptuses. Immunocytochemical staining was also used to localize trophoblast-specific and class I major histocompatibility complex (MHC) antigens on some specimens. Relaxin mRNA and relaxin were both present in the single-cell non-invasive trophoblast layer of the allantochorion between 45 and 153 days of gestation in all three types of equine pregnancy examined. Both, however, were absent from the invasive trophoblast cells of the progenitor chorionic girdle and the differentiated trophoblast cells of the endometrial cups throughout the latters' 60-80-day period of development and regression. Discrete and irregularly spaced clusters of elongated pseudostratified trophoblast cells on the allantochorion remained negative for relaxin mRNA and ligand, but stained strongly for equine trophoblast-specific antigens. These areolae-like structures of the mature horse placenta overlie the mouths of endometrial glands between adjacent microcotyledons and they are clearly involved with the uptake of uterine milk for fetal sustenance. It is speculated that their loose attachment to the endometrium and weak expression of class 1 MHC antigens may serve to tolerize the mother to the paternally-inherited histocompatibility antigens of the fetus.

Relaxin-like factor (RLF) mRNA expression in the fallow deer.

Employing RT- and RACE-PCR on RNA isolated from testicular tissue, we have cloned the coding cDNA sequence for the RLF, also known as Insl3, of the fallow deer. The RLF coding sequence consisted of 396 bp encoding a peptide of 131 amino acids and shared highest homology with bovine, sheep and goat RLF. Northern analysis revealed a single 0.9 kb transcript in the deer testis. There is only one RLF gene in the deer genome. Nonradioactive in situ hybridization revealed the Leydig cells to be the sole source for RLF mRNA in the deer testis. In the non-pregnant uterus, RLF transcripts were located in the luminal and glandular epithelium of the endometrium. Within the ovary of the pregnant doe, follicular theca interna cells and the corpus luteum expressed RLF transcripts. In uteroplacental tissues, luminal and glandular epithelium, fetal uninucleate and binucleate trophoblast cells (BNC) of the basic villous trophoblast layer expressed RLF mRNA. BNC located at the apical trophoblast layer or the tip of the fetal villus were devoid of RLF transcripts. Pseudostratified trophoblast cells at the base of fetal villi coexpressed RLF mRNA and immunoreactive MHC class Ib molecules.

Immunochemical mapping of gonadotropins.

As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.

Somatic isoform of angiotensin I-converting enzyme in the pathology of testicular germ cell tumors.

Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent seminomas (P =.032 for sACE; P =.005 for PIAP). Both sACE and PIAP often showed a decreased and more heterogeneous but still moderate expression in 91 classic seminomas (median IRS of 8) and were completely absent in tumor cells of spermatocytic seminomas. Despite all similarities, we found sACE and PIAP differently regulated during GCT progression. This was documented by a well-preserved expression of either sACE or PIAP or both in all classic seminomas, low PIAP immunoreactivity in metastasis of seminomas, and completely diverging expression patterns in nonseminomatous GCT. Our findings underline the close molecular relationship between IGCN and seminoma, and suggest sACE as an appropriate marker for seminomatous differentiated tumors. HUM PATHOL 31:1466-1476.

Identification and selective destruction of shared epitopes in human chorionic gonadotropin beta subunit.

The feasibility of producing epitope-specific antigens by mutation of the gene is demonstrated, the aim being to eliminate unwanted surface epitopes yet allowing the natural folding of the protein to maintain the desired epitope(s). The model protein is the beta subunit of human chorionic gonadotropin (hCG beta) which previously has been used as an immunological contraceptive vaccine but has extensive cross-reaction with human luteinizing hormone. Of a series of mutants made, the mutant with substitutions of Glu for Arg 68, Ser for Arg 74, His for Gly 75 and His for Val 79, lost the ability to react with a panel of cross-reacting monoclonal antibodies while retaining the discontinuous and linear epitopes specific to the holo-hormone. In addition, allocation of amino acid residues to established epitope clusters could be made: residues 24, 25, 68 and 71 probably contribute to the cluster termed beta 3, residues 20, 21, 22, 75 and 77 to cluster beta 6 and residue 68 to clusters beta 2, beta 4 and beta 5.

Relaxin-like factor expression in a human ovarian Sertoli-Leydig cell tumor.

Relaxin-like factor (RLF), a new member of the insulin-like growth factor family, is a reliable marker for normal Leydig cells in the human postpubertal testis (1). Expression of the RLF gene appears to be developmentally regulated, given that only during puberty is RLF expression up-regulated. We recently demonstrated down-regulation of the human RLF gene in testicular Leydig cell tumors indicating dedifferentiation of the Leydig cells within the tumor (2). Ovarian Sertoli-Leydig cell tumors (SLCTs), histologically typed as androblastomas, are rare, potentially malignant sex-cord stromal tumors exhibiting testicular-like structure and differentiation of various degrees. In the present study, we investigated the expression of RLF, 17alpha-hydroxylase, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), Ki-67, and cytokeratin 18 in a human ovarian SLCT of low differentiation.

Arylhydrocarbon receptor expression in the human endometrium.

OBJECTIVE: To determine the expression and localization of the arylhydrocarbon (dioxin) receptor in human endometrium throughout the normal menstrual cycle. DESIGN: Retrospective immunohistochemical and in situ hybridization study. SETTING: Academic research unit. PATIENT(S): Premenopausal women (n = 86), aged 25 to 45 years, with histologically normal endometrium undergoing curettage or hysterectomy. INTERVENTION(S): Endometrial samples were collected from days 3 to 26 of the cycle by superficial scrapings of the uterine cavity or by hysterectomy. MAIN OUTCOME MEASURE(S): Expression of arylhydrocarbon receptor mRNA and protein. RESULT(S): Arylhydrocarbon receptor was expressed in 43% of the endometria studied and was correlated with the day of the cycle. The maximum of immunopositive endometria was found around the time of ovulation. Immunostaining decreased with increasing age of the patients. The receptor protein was localized exclusively in the apical part of the cytoplasm in the epithelial cells of the endometrial glands. In women positive for arylhydrocarbon receptor, arylhydrocarbon receptor mRNA was expressed in the cytoplasm of endometrial epithelial cells. CONCLUSION(S): Our results describe the expression of the arylhydrocarbon receptor in human endometrium and indicate a possible involvement of this transcription factor in endometrial function in women during the reproductive phase.

Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest.

During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.

Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrial fibroblasts.

3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are cholesterol-lowering drugs that exert other cellular effects and underlie their beneficial health effects, including those associated with myocardial remodeling. We recently demonstrated that statins induces apoptosis and autophagy in human lung mesenchymal cells. Here, we extend our knowledge showing that statins simultaneously induces activation of the apoptosis, autophagy and the unfolded protein response (UPR) in primary human atrial fibroblasts (hATF). Thus we tested the degree to which coordination exists between signaling from mitochondria, endoplasmic reticulum and lysosomes during response to simvastatin exposure. Pharmacologic blockade of the activation of ER-dependent cysteine-dependent aspartate-directed protease (caspase)-4 and lysosomal cathepsin-B and -L significantly decreased simvastatin-induced cell death. Simvastatin altered total abundance and the mitochondrial fraction of proapoptotic and antiapoptotic proteins, while c-Jun N-terminal kinase/stress-activated protein kinase mediated effects on B-cell lymphoma 2 expression. Chemical inhibition of autophagy flux with bafilomycin-A1 augmented simvastatin-induced caspase activation, UPR and cell death. In mouse embryonic fibroblasts that are deficient in autophagy protein 5 and refractory to autophagy induction, caspase-7 and UPR were hyper-induced upon treatment with simvastatin. These data demonstrate that mevalonate cascade inhibition-induced death of hATF manifests from a complex mechanism involving co-regulation of apoptosis, autophagy and UPR. Furthermore, autophagy has a crucial role in determining the extent of ER stress, UPR and permissiveness of hATF to cell death induced by statins.

Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction.

The dioxin/aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsive to both natural and man-made environmental compounds. AhR and its nuclear partner ARNT are expressed in the female reproductive tract in a variety of species and several indications suggest that the AhR might play a pivotal role in the physiology of reproduction. Furthermore, it appears to be the mediator of most, if not all, the adverse effects on reproduction of a group of highly potent environmental pollutants collectively called aryl hydrocarbons (AHs), including the highly toxic compound 2,3,7,8-tetrachlor-odibenzo-p-dioxin (TCDD). Although a large body of recent literature has implicated AhR in multiple signal transduction pathways, the mechanisms of action resulting in a wide spectrum of effects on female reproduction are largely unknown. Here we summarize the major types of molecular cross-talks that have been identified for the AhR and linked cell signaling pathways and that are relevant for the understanding of the role of this transcription factor in female reproduction.