Benzo(alpha)pyrene-induced up-regulation of CYP1A2 gene expression: role of adrenoceptor-linked signaling pathways

CYP1A2, a principal catalyst for metabolism of various therapeutic drugs and carcinogens, among others, is in part regulated by the stress response. This study was designed to assess whether catecholamines and in particular adrenergic receptor-dependent pathways, modulate benzo(alpha)pyrene (B(alpha)P)-induced hepatic CYP1A2. To distinguish between the role of central and peripheral catecholamines in the regulation of CYP1A2 induction, the effect of central and peripheral catecholamine depletion using reserpine was compared to that of peripheral catecholamine depletion using guanethidine. The effects of peripheral adrenaline and L-DOPA administration were also assessed. The results suggest that alterations in central catecholamines modulate 7-methoxyresorufin O-demethylase activity (MROD), CYP1A2 mRNA and protein levels in the B(alpha)P-induced state. In particular, central catecholamine depletion, dexmedetomidine-induced inhibition of noradrenaline release and blockade of alpha(1)-adrenoceptors with prazosin, up-regulated CYP1A2 expression. Phenylephrine and dexmedetomidine-induced up-regulation may be mediated, in part, via peripheral alpha(1)- and alpha(2)-adrenoceptors, respectively. On the other hand, the L-DOPA-induced increase in central dopaminergic activity was not followed by any change in the up-regulation of CYP1A2 expression by B(alpha)P. Central noradrenergic systems appeared to counteract up-regulating factors, most likely via alpha(1)- and alpha(2)-adrenoceptors. In contrast, peripheral alpha- and beta-adrenoceptor-related signaling pathways are linked to up-regulating processes. The findings suggest that drugs that bind to adrenoceptors or affect central noradrenergic neurotransmission, as well as factors that challenge the adrenoceptor-linked signaling pathways may deregulate CYP1A2 induction. This, in turn, may result in drug-therapy and drug-toxicity complications.     

In vivo dynamics of Rac-membrane interactions

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.     

v-Src rescues actin-based cytoskeletal architecture and cell motility and induces enhanced anchorage independence during oncogenic transformation of focal adhesion kinase-null fibroblasts

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.     

Integrin signalling in directed cell migration

Migrating cells tend to continue moving in the same direction, a property called persistence. During migration, cells, by definition, form new adhesions at their front and break old adhesions at the rear. We hypothesize that the distinction between new adhesions at the front and older adhesions at the rear plays a major role in directional persistence. We propose specific mechanisms of persistence on the basis of known properties of integrin signals, in hope of stimulating investigation of these ideas.