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The Gadd45 family of proteins includes Gadd45␣, MyD118/Gadd45␤, and CR6/OIG37/Gadd45␥. These proteins play important roles in maintaining genomic stability and in regulating the cell cycle. This study reports the cloning of a novel protein called CR6-interacting factor 1 (CRIF1) which interacts with Gadd45␣, MyD118/Gadd45␤, and CR6/OIG37/Gadd45␥. CRIF1 binds specifically to the Gadd45 family proteins, as determined by an in vitro glutathione Stransferase pull-down assay and an in vivo mammalian cell two-hybrid assay along with coimmunoprecipitation assays. CRIF1 mRNA is highly expressed in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues. CRIF1 localizes exclusively to the nucleus and colocalizes with Gadd45␥. Recombinant CRIF1 inhibits the histone H1 kinase activity of immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E, and the inhibitory effects were additive with Gadd45 proteins. Overexpression of CRIF1 increases the percentage of cells in G 1 , decreases the percentage of cells in S phase, and suppresses growth in NIH3T3 cells. The downregulation of endogenous CRIF1 by the transfection of the small interfering RNA duplexes resulted in the inactivation of Rb by phosphorylation and decreased the G 1 phase cell populations. Expression of CRIF1 is barely detectable in adrenal adenoma and papillary thyroid cancer and much lower than in adjacent normal tissue. The results presented here suggest that CRIF1 is a novel nuclear protein that interacts with Gadd45 and may play a role in negative regulation of cell cycle progression and cell growth.

Curcumin, a natural compound, is a well-known chemopreventive agent with potent anticarcinogenic activity in a wide variety of tumor cells. Curcumin inhibits cancer cell proliferation in part by suppressing cyclin D1 and inducing expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 . Both p53-dependent and p53-independent mechanisms regulate p21Waf1/Cip1 expression, but the mechanism by which curcumin regulates p21Waf1/Cip1 expression remains unknown. Here, we report that transcription of the p21Waf1/Cip1 gene is activated by early growth response-1 (Egr-1) independently of p53 in response to curcumin treatment in U-87MG human glioblastoma cells. Egr-1 is a transcription factor that helps regulate differentiation, growth, and apoptosis in many cell types. Egr-1 expression is induced by curcumin through extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK), but not the p38, mitogen-activated protein kinase (MAPK) pathways, which mediate the transactivation of Elk-1. Transient expression of Egr-1 enhanced curcumin-induced p21 Waf1/Cip1 promoter activity, whereas suppression of Egr-1 expression by small interfering RNA abrogated the ability of curcumin to induce p21 Waf1/Cip1 promoter activity. In addition, stable knockdown of Egr-1 expression in U-87MG cells suppressed curcumin-induced p21 expression. Our results indicate that ERK and JNK MAPK/Elk-1/Egr-1 signal cascade is required for p53-independent transcriptional activation of p21 Waf1/Cip1 in response to curcumin in U-87MG human glioblastoma cells. [Cancer Res 2008;68(5):1369-77]

Recent evidence has shown that Ras homolog enriched in brain (Rheb) is dysregulated in Alzheimer's disease (AD) brains. However, it is still unclear whether Rheb activation contributes to the survival and protection of hippocampal neurons in the adult brain. To assess the effects of active Rheb in hippocampal neurons in vivo, we transfected neurons in the cornu ammonis 1 (CA1) region in normal adult rats with an adeno-associated virus containing the constitutively active human Rheb (hRheb(S16H)) and evaluated the effects on thrombin-induced neurotoxicity. Transduction with hRheb(S16H) significantly induced neurotrophic effects in hippocampal neurons through activation of mammalian target of rapamycin complex 1 (mTORC1) without side effects such as long-term potentiation impairment and seizures from the alteration of cytoarchitecture, and the expression of hRheb(S16H) prevented thrombin-induced neurodegeneration in vivo, an effect that was diminished by treatment with specific neutralizing antibodies against brain-derived neurotrophic factor (BDNF). In addition, our results showed that the basal mTORC1 activity might be insufficient to mediate the level of BDNF expression, but hRheb(S16H)-activated mTORC1 stimulated BDNF production in hippocampal neurons. These results suggest that viral vector transduction with hRheb(S16H) may have therapeutic value in the treatment of neurodegenerative diseases such as AD.

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