All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38␣/␤/␥ MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38␥ MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38␥ MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.

Cannabinoids are a group of compounds present in Cannabis plant (Cannabis sativa L.). They mediate their physiological and behavioral effects by activating specific cannabinoid receptors. With the recent discovery of the cannabinoid receptors (CB1 and CB2) and the endocannabinoid system, research in this field has expanded exponentially. Cannabinoids have been shown to act as potent immunosuppressive and anti-inflammatory agents and have been shown to mediate beneficial effects in a wide range of immune-mediated diseases such as multiple sclerosis, diabetes, septic shock, rheumatoid arthritis, and allergic asthma. Cannabinoid receptor 1 (CB1) is mainly expressed on the cells of the central nervous system as well as in the periphery. In contrast, cannabinoid receptor 2 (CB2) is predominantly expressed on immune cells. The precise mechanisms through which cannabinoids mediate immunosuppression is only now beginning to be understood and can be broadly categorized into four pathways: apoptosis, inhibition of proliferation, suppression of cytokine and chemokine production and induction of T regulatory cells (T regs). Studies from our laboratory have focused on mechanisms of apoptosis induction by natural and synthetic cannabinoids through activation of CB2 receptors. In this review, we will focus on apoptotic mechanisms of immunosuppression mediated by cannabinoids on different immune cell populations and discuss how activation of CB2 provides a novel therapeutic modality against inflammatory and autoimmune diseases as well as malignancies of the immune system, without exerting the untoward psychotropic effects.

Tissue transglutaminase or type II transglutaminase (TGase) 1 is an 87-kDa protein that contains two key catalytic activities, the ability to catalyze protein-amine cross-links and a GTP binding and hydrolysis activity. The transamidation reaction of TGase has been well studied and consists of the Ca 2ϩ -dependent formation of covalent bonds between the ␥-carboxamide groups of peptide-bound glutamine residues and the primary amino groups of a wide variety of proteins (1, 2). Transamidation has been implicated in a number of biological processes such as axonal regeneration, cellular differentiation, and apoptosis (3-12). Many of the early studies of TGase have identified it as being present in cells and tissues undergoing apoptosis (7-10, 13, 14) and implicated the transamidation activity of TGase as a potentiator of programmed cell death (15,16). However, there is growing evidence that TGase may not directly mediate apoptosis. TGase Ϫ/Ϫ mice showed no major developmental abnormalities, and the thymocytes from these mice were no less susceptible to apoptosis than TGase ϩ/ϩ cells (17,18). Studies examining the role of TGase in retinoic acid (RA)-mediated signaling (12), as well as studies demonstrating that TGase was required for neurite outgrowth (19), suggest that TGase may exhibit protective effects against apoptotic signals. It has been well documented that RA up-regulates both the expression and transamidation activity of TGase (12, 20 -22). We have recently shown that the ability of RA to up-regulate TGase expression and activity is required for the ability of RA to protect against apoptosis induced by a synthetic retinoid analog, all-trans-N-(4-hydroxyphenyl)retinamide (HPR) (12). The TGase-mediated inhibition of apoptosis appeared to involve its transamidation activity and its ability to bind GTP (12), a result that may suggest a role for TGase in more than one anti-apoptotic signaling pathway.To understand how TGase is acting as an anti-apoptotic factor, we set out to investigate substrates of TGase that are potential regulators of cell death. One such protein was the retinoblastoma gene product (Rb), a well established regulator of the G 1 /S checkpoint in the cell cycle (23)(24)(25). In addition to its role in cell cycle regulation and cellular differentiation, Rb has also been implicated in apoptosis (26). Rb Ϫ/Ϫ mice are embryonic lethal with significant apoptosis occurring in the developing nervous system and lens of the eye (27-30). The tumor suppressor gene p53 activates a pathway in response to various cellular insults that results in the degradation of Rb (31), a critical event that shifts cells toward apoptosis. The Rb protein was initially identified as a substrate for TGase-mediated transamidation in U937 cells in early stages of apoptosis (11). This finding, when combined with other results showing increased expression of TGase in apoptotic cells (7-10, 13, 14), led to the suggestion that TGase was a pro-apoptotic protein. However, we have found that TGase acts in a protective fashion (12) b...

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPRinduced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.

The primary aim of these studies was to identify and biochemically characterize GTP-binding proteins in the nucleus. We found that an 80 kDa protein was responsible for the majority of the GTP-binding activity detected in rabbit liver nuclear preparations as assayed by photoaffinity labeling with [alpha-32P]GTP. The GTP-binding activity was partially extracted only after treatment of nuclear envelope preparations with 0.5 M NaCl and 1% Triton-X 100, which suggested that this GTP-binding protein was a component of the nuclear pore/lamina fraction. The Triton-X-100/NaCl-solubilized 80 kDa protein was purified by a series of steps that included DEAE-Sephacel, Mono-Q, and Ultrogel AcA34 chromatographies. Microsequence analysis of two peptides generated by trypsin digestion of the 80 kDa protein indicates that it shares sequence similarity with the tissue transglutaminases. Purified preparations of the 80 kDa protein show a Ca(2+)-stimulated transglutaminase activity, as assayed by the incorporation of [3H]putrescine into caesin, which is strongly inhibited by GTP but not by GDP. A 36 kDa GTP-binding protein copurified with the 80 kDa GTP-binding protein through all of the chromatography steps and sequence analysis suggests that the 36 kDa protein represents a proteolytic fragment of the amino-terminal half of the 80 kDa protein and thus serves to mark the GTP-binding domain within the 80 kDa protein. The 36 kDa fragment has a significantly higher efficiency of [alpha-32P]GTP incorporation compared to the 80 kDa protein, suggesting that the carboxyl-terminal half of the GTP-binding protein/transglutaminase imparts a negative constraint on GTP-binding activity or on the subsequent incorporation of radiolabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)