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Iron (Fe) plays an important role in proliferation, and Fe deficiency results in G 1 /S arrest. Despite this, the precise role of Fe in cell-cycle control remains unclear. Cyclin D1 plays a critical function in G 1 progression by interacting with cyclindependent kinases. Previously, we examined the effect of Fe depletion on the expression of cell-cycle control molecules and identified a marked decrease in cyclin D1 protein, although the mechanism involved was unknown. In this study, we showed that cyclin D1 was regulated posttranscriptionally by Fe depletion. Iron chelation of cells in culture using desferrioxamine (DFO) or 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) decreased cyclin D1 protein levels after 14 hours and was rescued by the addition of Fe. Cyclin D1 half-life in control cells was 80 ؎ 15 minutes (n ‫؍‬ 5), while in chelator-treated cells it was significantly (P < .008) decreased to 38 ؎ 3 minutes (n ‫؍‬ 5). Proteasomal inhibitors rescued the Fe chelator-mediated decrease in cyclin D1 protein, suggesting the role of the proteasome. In Fe-replete cells, cyclin D1 was degraded in an ubiquitin-dependent manner, while Fe depletion induced a ubiquitin-independent pathway. This is the first report linking Fe depletionmediated growth suppression at G 1 /S to a mechanism inducing cyclin D1 proteolysis. IntroductionProliferation is dependent upon iron (Fe). [1][2][3][4] The highest demand for Fe occurs during the late G 1 and S phases of the cell cycle, 5,6 due in part to the activity of the Fe-requiring enzyme of DNA synthesis, ribonucleotide reductase (RR). 5,6 Iron-deprivation inhibits RR activity and results in G 1 /S arrest. 7 However, little is known at the molecular level concerning the role of Fe in cell-cycle progression.Cell-cycle progression is controlled in part by the cyclins, cyclin-dependent kinases (cdk's), and cyclin-dependent kinase inhibitors (cdki's). 7,8 Cyclins interact with cdk's to form catalytically active heterodimers. 8 Fe depletion decreases cyclin D1 (CD1) protein expression. 9,10 However, the mechanism responsible remains unknown. CD1 assembles with cdk-4 or cdk-6, generating an active holoenzyme that catalyzes a rate-limiting step in G 1 /S progression. 7,8 This complex phosphorylates the retinoblastoma protein which regulates S-phase entrance. 8 Overexpression of CD1 releases cells from their normal controls and acts as an oncogene. 11,12 In fact, pharmacologic targeting of CD1 may lead to novel antitumor agents. 13 CD1 is regulated by changes in transcription, protein stability, mRNA turnover, and nucleocytoplasmic transport of the transcript and protein. [14][15][16][17][18][19] CD1 protein expression can be regulated by degradation via the ubiquitin-dependent 20 or ubiquitin-independent 21 proteasome pathway. Phosphorylation of CD1 at Thr-286 by glycogen synthase kinase-3␤ (GSK-3␤) can result in ubiquitination and proteasomal degradation. 14,16 However, a GSK-3␤-independent pathway of proteasomal CD1 degradation has been reported. 17 CD1 can also be targete...

This study describes a unique function of taurocholate in bile canalicular formation involving signaling through a cAMP-Epac-MEK-Rap1-LKB1-AMPK pathway. In rat hepatocyte sandwich cultures, polarization was manifested by sequential progression of bile canaliculi from small structures to a fully branched network. Taurocholate accelerated canalicular network formation and concomitantly increased cAMP, which were prevented by adenyl cyclase inhibitor. The cAMP-dependent PKA inhibitor did not prevent the taurocholate effect. In contrast, activation of Epac, another cAMP downstream kinase, accelerated canalicular network formation similar to the effect of taurocholate. Inhibition of Epac downstream targets, Rap1 and MEK, blocked the taurocholate effect. Taurocholate rapidly activated MEK, LKB1, and AMPK, which were prevented by inhibition of adenyl cyclase or MEK. Our previous study showed that activated-LKB1 and AMPK participate in canalicular network formation. Linkage between bile acid synthesis, hepatocyte polarization, and regulation of energy metabolism is likely important in normal hepatocyte development and disease.primary hepatocytes | occludin | P-glycoprotein H epatocytes, the major epithelial cells in the liver, are polarized. Tight junction proteins, including occludin, claudin, and ZO-1, seal the canalicular lumen, thereby separating apical and basolateral membrane domains and forming the bile canaliculus (1). Polarization is essential for biliary secretion. The mechanisms of polarization are complex and include cytoskeletal, tight junctional, and intracellular trafficking components (2-5). Loss of polarity causes bile secretory failure (cholestasis) and liver damage (6).Bile acids are synthesized from cholesterol in hepatocytes, secreted by ABCB11 (Bsep) into the bile canaliculus, and then mostly absorbed into the enterohepatic circulation (7). The major bile acids in mammals are tauro or glycine conjugates of cholic, deoxycholic, and chenodeoxycholic acids (8). In addition to their traditional function in emulsification of dietary fat (8), recent studies reveal that bile acids function as signaling molecules (9), which increase calcium mobilization (10) and cellular cAMP (9); translocate and activate protein kinase C (11), nuclear farnesoid X receptor (FXR), and pregnane X receptors (PXR) (7, 9); activate PI3K/AKT/glycogen synthase kinase 3 (GSK3) (12); and enhance liver regeneration (13).During embryonic development, early fetal hepatocytes are not polarized (14-16). In fetal mice and rats, infrequent small canaliculi are present, but do not attain an adult appearance until several days postpartum (17). Bile acid synthesis, turnover, and secretion are sparse in fetal liver, and rapidly increase postnatally (18,19), concomitant with hepatocyte polarization and development of a branched canalicular network. Based on these events, we postulated that bile acids may regulate hepatocyte polarization and canalicular formation. Using collagen sandwich cultures of rat primary hepatocytes, we confirmed this hy...