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-κB in TNFα-stimulated cells and has been shown to be activated in response to interleukin-1β (IL-1β). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-κB 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-κB activation following IL-1β stimulation. Together, these results indicate that p62 is an important intermediary in the NF-κB activation pathways implemented through non-degradative ubiquitination events.

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.

The pro-inflammatory cytokine tumor necrosis factor (TNF) α signals both cell survival and death. The biological outcome of TNFα treatment is determined by the balance between survival factors and Jun NH(2)-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 TNFα stimulation. We also show that TRAF6 and TRAF7 promote unconventional polyubiquitination of the anti-apoptotic protein c-FLIP(L) and demonstrate that degradation of c-FLIP(L) also occurs through a lysosomal pathway. RNA interference-mediated depletion of TRAF7 correlates with increased c-FLIP(L) expression level, which, in turn, results in resistance to TNFα cytotoxicity. Collectively, our results indicate an important role for TRAF7 in the activation of JNK following TNFα 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.

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 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.