Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-cB in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-KcB by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose-and time-dependent manner. Besides TNF, CAPE also inhibited NF-cB activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-KcB activation. CAPE prevented the translocation of the p65 subunit of NF-cB to the nucleus and had no significant effect on TNF-induced IKcBa degradation, but did delay IucBa resynthesis. The effect of CAPE on inhibition of NF-cB binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-i, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-KcB activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

Birt-Hogg-Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1͞2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5 AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt-Hogg-Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and͞or nutrient sensing through the AMPK and mTOR signaling pathways.hamartoma syndrome ͉ renal cancer ͉ Birt-Hogg-Dubé ͉ tumor suppressor B irt-Hogg-Dubé (BHD) syndrome predisposes patients to develop hair follicle hamartomas, lung cysts, and an increased risk for renal neoplasia (1-3). BHD patients develop bilateral, multifocal renal tumors with a variety of histologies (4). We mapped the BHD locus to chromosome 17p11.2 by linkage analysis in BHD kindreds (5, 6) and identified germ-line mutations in a gene with unknown function that is highly conserved (7,8). Twenty-two unique mutations predicted to truncate the BHD protein folliculin (FLCN), including a ''hot spot'' insertion͞deletion in a C 8 tract, were identified in 84% of BHD kindreds (9). Somatic ''second-hit'' mutations identified in BHD-associated renal tumors suggest a tumor-suppressor function for FLCN (10), underscored by loss of BHD mRNA expression in renal tumors from BHD patients (11).Recent studies suggest that several hamartoma syndromes may be linked through the convergent energy͞nutrient-sensing pathways involved in mammalian target of rapamycin (mTOR) regulation (12-15). These inherited syndromes are characterized by multiple hamartomas and an increased risk of cancer. Germ-line mutations have been identified in four causative genes: LKB1, responsible for Peutz-Jeghers syndrome (16-18), TSC1 and TSC2, responsible for tuberous sclerosis complex (TSC) (19), and PTEN, responsible for Cowden syndrome (20). Loss of gene function leads to dysregulation of mTOR, which regulates cell growth and size through stimulation of protein synthesis (15, 21, 22).BHD syndrome, also a hamartoma disorder, displays phenotypic similarities to TSC that have led to speculation that BHD may function in the pathway(s) signaling through mTOR (12,23). To ascertain FLCN function,...