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Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Background: The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor is a key regulator of cell survival, proliferation, and gene expression. Although activat...

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in ‘de novo lipogenesis’ and β-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

The nuclear factor (NF)-κB signaling pathway controls a variety of important biological functions, including immune and inflammatory responses, differentiation, cell growth, tumorigenesis, and apoptosis. Two distinct pathways of NF-κB activation are known. The classical, canonical pathway is found virtually in all mammalian cells and NF-κB activation is mediated by the IKK complex, consisting of the IKK1/IKKα and IKK2/IKKβ catalytic kinase subunits and the NF-κB essential modulator (NEMO)/IKKγ protein. The NF-κB-driven transcriptional responses to many different stimuli have been widely characterized in the pathophysiology of the mammalian immune system, mainly because this transcription factor regulates the expression of cytokines, growth factors, and effector enzymes in response to ligation of cellular receptors involved in immunity and inflammation. However, an impressive literature produced in the last two decades shows that NF-κB signaling plays an important role also outside of the immune system, performing different roles and functions depending on the type of tissue and organ. In thyroid, NF-κB signaling is crucial for thyrocytes survival and expression of critical thyroid markers, including Nis, Ttf1, Pax8, Tpo, and thyroglobulin, making this transcription factor essential for maintenance of normal thyroid function.

The nuclear factor (NF)-κB signaling pathway controls a variety of important biological functions, including immune and inflammatory responses, differentiation, cell growth, tumorigenesis, and apoptosis. Two distinct pathways of NF-κB activation are known. The classical, canonical pathway is found virtually in all mammalian cells and NF-κB activation is mediated by the IKK complex, consisting of the IKK1/IKKα and IKK2/IKKβ catalytic kinase subunits and the NF-κB essential modulator (NEMO)/IKKγ protein. The NF-κB-driven transcriptional responses to many different stimuli have been widely characterized in the pathophysiology of the mammalian immune system, mainly because this transcription factor regulates the expression of cytokines, growth factors, and effector enzymes in response to ligation of cellular receptors involved in immunity and inflammation. However, an impressive literature produced in the last two decades shows that NF-κB signaling plays an important role also outside of the immune system, performing different roles and functions depending on the type of tissue and organ. In thyroid, NF-κB signaling is crucial for thyrocytes survival and expression of critical thyroid markers, including Nis, Ttf1, Pax8, Tpo, and thyroglobulin, making this transcription factor essential for maintenance of normal thyroid function.

Human BCL10 (hBCL10) protein is a signal transduction molecule originally identified because of its direct involvement in a subset of mucosa-associated lymphoid tissue (MALT) lymphomas, and later recognized as a crucial factor in regulating activation of NF-kB transcription factor following antigen receptor stimulation on lymphocytes. In this study, we characterized the NF-kB inducing activity of porcine BCL10 (pBCL10). pBCL10 oligimerizes, binds to components of the CARMA/ BCL10/MALT1 complex and forms cytoplasmic filaments. Functionally, in human cells pBCL10 is more effective in activating NF-kB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that pBcl10 can functionally replace the human protein and retains its higher NF-kB-inducing property in the absence of hBCL10. Our results contribute useful information on BCL10 protein in pigs, and may help the development of strategies based on the control of the immune response in pigs.

Genetic and environmental factors contribute to thyroid diseases. Although still debated, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to induce thyroid dysfunction in humans and rodents. The data here reported point out the contribution of the exposure window and genetic background in mediating the low-dose TCDD effects on thyroid. Indeed, early (from E0.5 to PND30) and low-dose (0,001 μg/kg/day) TCDD exposure reduced the circulating fT4 and altered the expression of thyroid specific transcripts. The role of genetic components was estimated monitoring the same markers in Pax8+/− and Nkx2-1+/− mice, susceptible to thyroid dysfunction, exposed to 0, 1 μg/kg/day TCDD from E15.5 to PND60. Haploinsufficiency of either Pax8 or Nkx2-1 genes exacerbated the effects of the exposure impairing the thyroid enriched mRNAs in sex dependent manner. Such effect was mediated by mechanisms involving the Nkx2-1/p53/p65/IĸBα pathway in vitro and in vivo. Foetal exposure to TCDD impaired both thyroid function and genes expression while thyroid development and differentiation did not appear significantly affected. In mouse, stronger effects were related to earlier exposure or specific genetic background such as either Pax8 or Nkx2-1 haploinsufficiency, both associated to hypothyroidism in humans. Furthermore, our data underline that long exposure time are needed to model in vitro and in vivo results.

The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.

The atypical protein kinase C-interacting protein p62/sequestosome-1 (p62) has emerged as a crucial molecule in a variety of cellular functions due to its involvement in various signaling mechanisms. p62 has been implicated in the activation of NF-?B in TNF?-stimulated cells and has been shown to be activated in response to interleukin-1? (IL-1?). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-?B transcription factor. Depletion of p62 obtained through a short interfering RNA targeting p62 mRNA abrogated TRAF6 capacity to promote NEMO ubiquitination and severely impairs NF-?B activation following IL-1? stimulation.Together, these results indicate that p62 is an important intermediary in the NF-?B activation pathways implemented through non-degradative ubiquitination events. Telethon Grant GGP08125B Scopus

The I-κB kinase (IKK) subunit NEMO/IKKγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology. The I-κB kinase (IKK) subunit NEMO/IKKoγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology.

Background: The CARMA-BCL10-MALT1 complex recruits signaling components leading to activation of NF-κB transcription factor. Results: The Rho guanine nucleotide exchange factor Net1 binds to BCL10, CARMA1, and CARMA3. Conclusion: Net1 modulates activation of NF-κB transcription factor. Significance: The results shown are important to understand events that control NF-κB activation and the mechanisms by which Net1 promotes cellular transformation., The molecular complexes containing CARMA proteins have been recently identified as a key components in the signal transduction pathways that regulate activation of nuclear factor κB (NF-κB) transcription factor. Here, we used immunoprecipitation coupled with mass spectrometry to identify cellular binding partners of CARMA proteins. Our data indicate that the Rho guanine nucleotide exchange factor Net1 binds to CARMA1 and CARMA3 in resting and activated cells. Net1 expression induces NF-κB activation and cooperates with BCL10 and CARMA proteins in inducing NF-κB activity. Conversely, shRNA-mediated abrogation of Net1 results in impaired NF-κB activation following stimuli that require correct CARMA-BCL10-MALT1 complex formation and functioning. Microarray expression data are consistent with a positive role for Net1 on NF-κB activation. Thus, this study identifies Net1 as a CARMA-interacting molecule and brings important information on the molecular mechanisms that control NF-κB transcriptional activity.

The complexes formed by BCL10, MALT1 and specific members of the family of CARMA proteins (CBM complex), have recently focused much attention because they represent a central hub regulating activation of the transcription factor NF-κB following various cellular stimulations. In this manuscript, we report the functional characterization of a Danio rerio 241 amino acids polypeptide ortholog of the Caspase recruiting domain (CARD)-containing protein BCL10. Biochemical studies show that zebrafish Bcl10 (zBcl10) dimerizes and binds to components of the CBM complex. Fluorescence microscopy observations demonstrate that zBcl10 forms cytoplasmic filaments similar to that formed by human BCL10 (hBCL10). Functionally, in human cells zBcl10 is more effective in activating NF-κB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that zBcl10 can functionally replace the human protein. Finally, we show that the zebrafish cell line PAC2 is suitable to carry out reporter assays for monitoring the activation state of NF- kB transcription factor. In conclusion, this work shows that zebrafish may excellently serve as a model organism to study complex and intricate signal transduction pathways, such as those that control NF-κB activation.

The pro-inflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNF alpha treatment is determined by the balance between survival factors and Jun NH2-terminal kinase (JNK) signaling, which promotes cell death. Here, we show that TRAF7, the most recently identified member of the TNF receptor-associated factors (TRAFs) family of proteins, is essential for activation of JNK following TNF alpha stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the antiapoptotic protein c-FLIPL and demonstrate that degradation of c-FLIPL also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIPL expression level, which, in turn, results in resistance to TNF alpha cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNF alpha stimulation and clearly point to an involvement of this protein in regulating the turnover of c-FLIP and, consequently, cell death.

The molecular complexes containing BCL10, MALT1 and CARMA proteins (CBM complex) have been recently identified as a key component in the signal transduction pathways that regulate activation of Nuclear Factor kappaB (NF-κB) transcription factor. Herein we identified the DEP domain-containing protein DEPDC7 as cellular binding partners of CARMA2 and CARMA3 proteins. DEPDC7 displays a cytosolic distribution and its expression induces NF-κB activation. Conversely, shRNA-mediated abrogation of DEPDC7 results in impaired NF-κB activation following G protein-coupled receptors stimulation, or stimuli that require CARMA2 and CARMA3, but not CARMA1. Thus, this study identifies DEPDC7 as a CARMA interacting molecule, and provides evidence that DEPDC7 may be required to specifically convey on the CBM complex signals coming from activated G protein-coupled receptors.