GLI1, a crucial mediator of sonic hedgehog signaling in prostate cancer, functions as a negative modulator for androgen receptor

Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from an androgen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.     

PINOID functions in root phototropism as a negative regulator

The PINOID (PID) family, which belongs to AGCVIII kinases, is known to be involved in the regulation of auxin efflux transporter PIN-FORMED (PIN) proteins through changes in the phosphorylation status. Recently, we demonstrated that the PID family is necessary for phytochrome-mediated phototropic enhancement in Arabidopsis hypocotyls and that the downregulation of PID expression by red-light pretreatment results in the promotion of the PIN-mediated auxin gradient during phototropic responses. However, whether PID participates in root phototropism in Arabidopsis seedlings has not been well studied. Here, we demonstrated that negative root phototropic responses are enhanced in the pid quadruple mutant and are severely impaired in transgenic plants expressing PID constitutively. The results indicate that the PID family functions in a negative root phototropism as a negative regulator. On the other hand, analysis with PID fused to a yellow fluorescent protein, VENUS, showed that unilateral blue-light irradiation causes a lower accumulation of PID proteins on the shaded side than on the irradiated side. This result suggests that the blue-light-mediated asymmetrical distribution of PID proteins may be one of the critical responses in phototropin-mediated signals during a negative root phototropism. Alternatively, such a transverse gradient of PID proteins may result from gravitropic stimulation produced by phototropic bending.     

Interaction of the receptor FGFRL1 with the negative regulator Spred1

FGFRL1 is a member of the fibroblast growth factor receptor family. It plays an essential role during branching morphogenesis of the metanephric kidneys, as mice with a targeted deletion of the Fgfrl1 gene show severe kidney dysplasia. Here we used the yeast two-hybrid system to demonstrate that FGFRL1 binds with its C-terminal, histidine-rich domain to Spred1 and to other proteins of the Sprouty/Spred family. Members of this family are known to act as negative regulators of the Ras/Raf/Erk signaling pathway. Truncation experiments further showed that FGFRL1 interacts with the SPR domain of Spred1, a domain that is shared by all members of the Sprouty/Spred family. The interaction could be verified by coprecipitation of the interaction partners from solution and by codistribution at the cell membrane of COS1 and HEK293 cells. Interestingly, Spred1 increased the retention time of FGFRL1 at the plasma membrane where the receptor might interact with ligands. FGFRL1 and members of the Sprouty/Spred family belong to the FGF synexpression group, which also includes FGF3, FGF8, Sef and Isthmin. It is conceivable that FGFRL1, Sef and some Sprouty/Spred proteins work in concert to control growth factor signaling during branching morphogenesis of the kidneys and other organs.     

C-reactive protein functions as a negative regulator of macrophage activation induced by apoptotic DNA

C-reactive protein (CRP), an acute-phase protein with an ability to bind to nuclear antigen, has been reported to regulate cytokine secretion and modulate immune responses. We previously reported that activated syngeneic lymphocyte-derived apoptotic DNA (apopDNA) could induce macrophage activation and contribute to the initiation and progression of lupus nephritis. It is reasonable to hypothesize that CRP might regulate apopDNA-induced macrophage activation. Herein, CRP was shown to promote macrophage-mediated apopDNA uptake by binding to apopDNA (CRP/apopDNA complex). Notably, CRP/apopDNA treatment inhibited the production of inflammatory cytokines and chemokines by macrophages which could be induced by apopDNA alone. Further coculture and transwell studies revealed that CRP/apopDNA-induced macrophages prohibited apopDNA-induced macrophage activation in an IL-10 dependent manner. These results provide insight into the potential mechanism of CRP regulatory activity in macrophage activation induced by apopDNA in the context of lupus nephritis and other autoimmune diseases.     

Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carboxyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.