T cell receptor (TCR) induces a series of signaling cascades and leads to activation of multiple transcription factors, including NF-B. Although the mechanism of TCR-induced NF-B activation is not fully understood, recent studies indicate that Bcl10 and CARMA1, two adaptor/scaffold proteins, play essential roles in mediating TCR-induced NF-B activation. MALT1/paracaspase is a caspase-like protein that contains an Nterminal death domain, two Ig-like domains, and a C-terminal caspase-like domain. It binds to Bcl10 through its Ig-like domains and cooperates with Bcl10 to activate NF-B. Recently, it has been shown that MALT1 is involved in mediating TCR signal transduction, leading to activation of NF-B. In this study, we show that MALT1 is recruited into the lipid rafts of the immunological synapse following activation of the TCR and the CD28 coreceptor (CD3/CD28 costimulation). This recruitment of MALT1 is dependent on CARMA1 because CD3/CD28 costimulation failed to recruit MALT1 into lipid rafts in CARMA1-deficient T cells. In addition, we also found that MALT1 not only binds to Bcl10 directly, but also associates with CARMA1 in a Bcl10-independent manner. Therefore, MALT1, Bcl10, and CARMA1 form a trimolecular complex. Expression of a MALT1 deletion mutant containing only the N-terminal death domain and the two Ig-like domains completely blocked CD3/CD28 costimulation-induced, but not tumor necrosis factor-␣-induced, NF-B activation. Together, these results indicate that MALT1 is a crucial signaling component in the TCR signaling pathway.T cell activation plays a critical role in regulation of the immune response. Dysregulation of this process results in cancer, autoimmunity, and immunodeficiency diseases. T cell activation is initiated by major histocompatibility complex molecules on antigen-presenting cells presenting antigen peptides to T cell receptors (TCRs) 1 on the surface of T cells. This stimulation of TCR-CD3 complexes induces a series of signal transduction events leading to activation of multiple transcription factors such as AP-1, NF-AT, and NF-B (1-3). However, stimulation of TCR/CD3 complexes alone is insufficient to activate T cells. Instead, this signal will induce T cell anergy or apoptosis. The complete activation of T cells requires a second "costimulatory" signal generated through interactions between B7-1(CD80)/B7-2(CD86) molecules on antigen-presenting cells and CD28 receptors on T cells. This combined stimulation of TCR/CD3 and CD28 molecules (known as CD3/CD28 costimulation) leads to optimal activation of multiple transcription factors, including NF-B, and subsequent production of interleukin-2 and other cytokines (4). The NF-B family of transcription factors plays a critical role in controlling expression of various cytokine and anti-apoptotic genes. NF-B activity is regulated through interactions with a series of cytoplasmic inhibitory proteins termed IB, which mask the nuclear localization signal of NF-B, thereby sequestering NF-B in the cytoplasm. Treatment of cells with various stim...

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer. Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation. Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B). Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold. Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1). Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog. In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881. Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment. This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.

BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.

T lymphocyte (T cell) activation and proliferation is induced by the activation of multiple signal transduction pathways. Earlier studies indicate that CARMA1, a Caspase Recruitment Domain (CARD) and Membrane-associated GUanylate Kinase domain (MAGUK)-containing scaffold protein, plays an essential role in NF-B activation induced by the costimulation of T cell receptor (TCR) and CD28 molecules. However, the molecular mechanism by which CARMA1 mediates TCR-CD28 costimulation-induced NF-B activation is not fully understood. Here we show that CARMA1 is constitutively oligomerized. This oligomerization of CARMA1 is through its Coiled-coil domain. Disruption of the predicted structure of the Coiled-coil domain of CARMA1 impaired its oligomerization and, importantly, abrogated CARMA1-mediated NF-B activation. Interestingly, disruption of the CC1 domain abrogates CARMA1 localization, whereas disruption of the CC2 domain seems to inhibit CARMA1 self-association. Together, our results demonstrate that the oligomerization of CARMA1 is required for TCR-induced NF-B activation.