We previously found that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway. Recently, it has been reported that mRNA for the water channel, ...aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected anN-glycosylated band of ∼34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt2cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt2cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt2cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.
Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We ...recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (
e.g., 102% with 1 μmol/L glucagon) and a simultaneous decrease in intracellular membranes (
e.g., 38% with 1 μmol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion. (H
epatology 2003;37:1435-1441.)
In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2
WS25/−
mouse), we have studied ...hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including
sec63
,
prkcsh
, and
pkd1a
. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide 1 μM or vitamin K3 100 μM and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against
sec63
,
prkcsh
, and
pkd1a
eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.
Abstract
Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. CCA is an uncommon, but devastating cancer that is increasing in ...incidence. Over the past 3 decades, 5-years survival rates have remained at 10%. Although surgical resection and liver transplantation are potentially curative therapies, most patients are diagnosed at late stages and are not eligible for these options. Therefore, it is imperative to identify novel targets leading to new therapeutic strategies for this devastating disease.
Cholangiocytes express primary cilia that function as chemo, mechano, and osmosensors controlling several molecular pathways. We showed cilia are absent in CCA cells, and experimental deciliation of normal cholangiocytes induced a malignant like phenotype, with significant invasion and proliferation, suggesting the loss of cilia could be associated with CCA development. LKB1 is a tumor suppressor described to be expressed in primary cilia in MDCK cells, and is involved in AMPK activation through a ciliary dependent mechanism. AMPK functions as metabolic and stress check points. Interestingly, patients with intrahepatic CCA and low expression of LKB1 have poor prognosis. Therefore, we hypothesized that primary cilia function as tumor suppressor organelles through a LKB1-AMPK-p53 pathway. To test this hypothesis, first LKB1 subcellular localization was evaluated by confocal microscopy. We show for the first time that LKB1 is enriched in cholangiocyte primary cilia in a normal human cholangiocyte cell line (NHC). However, in experimental deciliated NHC and the CCA cell line KMCH, LKB1 is found dispersed in the cell cytoplasm. Next, we analyzed cellular expression levels of LKB1, AMPK (T172), p53 (s15), p53 and p21 by western blot. We found experimentally deciliated cholangiocytes and CCA cells exhibit defective AMPK signaling characterized by lower levels of LKB1 (-70% and -85%), AMPK (T172) (-72% and -71%), p53 (-37% and -83%), p53 (S15) (-38% and -100%) and p21 (-59% and -87%) relative to NHC. These molecular characteristics correlated with increased cell proliferation in deciliated cells (28%) and CCA cells (27%). Finally, we attempted to rescue the phenotype by using the AMPK activator, ampkinone. This treatment induced 20% inhibition of proliferation on experimental deciliated cholangiocytes and 18% in CCA cells, while not affecting NHC cells and the inhibition was associated with phosphorylation of AMPK and p21 up-regulation. In summary, our data suggest cholangiocyte primary cilia may normally function as tumor suppressors via a mechanism involving LKB1 and AMPK. The loss of cilia in CCA impairs LKB1-AMPK-p53-p21 signaling inducing a proliferative phenotype that may be rescued by specific activation of AMPK, warranting further studies to assess its use as a potential therapeutic approach.
Citation Format: Adrian P. Mansini, Kristen M. Thelen, Sergio A. Gradilone. Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2017-361
Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Santa Fe, ...Argentina
Submitted 18 September 2008
; accepted in final form 28 January 2009
Glucagon stimulates the vesicle trafficking of aquaporin-8 (AQP8) water channels to the rat hepatocyte canalicular membranes, a process thought to be relevant to glucagon-induced bile secretion. In this study, we investigated whether glucagon is able to modulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100 g body wt ip in 2, 3, or 6 equally spaced doses for 8, 16, and 36 h, respectively. Immunoblotting analysis showed that hepatic 34-kDa AQP8 was significantly increased by 79 and 107% at 16 and 36 h, respectively. Hepatic AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real-time PCR, was not modified over time, suggesting a posttranscriptional mechanism of AQP8 protein increase. Glucagon effects on AQP8 were directly studied in primary cultured rat hepatocytes. Immunoblotting and confocal immunofluorescence microscopy confirmed the specific glucagon-induced AQP8 upregulation. The RNA polymerase II inhibitor actinomycin D was unable to prevent glucagon effect, providing additional support to the nontranscriptional upregulation of AQP8. Cycloheximide also showed no effect, suggesting that glucagon-induced AQP8 expression does not depend on protein synthesis but rather on protein degradation. Inhibitory experiments suggest that a reduced calpain-mediated AQP8 proteolysis could be involved. The action of glucagon on hepatocyte AQP8 was mimicked by dibutyryl cAMP and suppressed by PKA or phosphatidylinositol-3-kinase (PI3K) inhibitors. In conclusion, our data suggest that glucagon induces the gene expression of rat hepatocyte AQP8 by reducing its degradation, a process that involves cAMP-PKA and PI3K signal pathways.
aquaporins; protein kinase A; phosphatidylinositol-3-kinase; calpains; liver
Address for reprint requests and other correspondence: R. A. Marinelli, Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR, Suipacha 570, 2000 Rosario, Santa Fe, Argentina (e-mail: rmarinel@unr.edu.ar)
Aquaporins (AQPs) are a family of water-selective channels that provide a major pathway for osmotically driven water transport through cell membranes. Some members of the aquaporin family have been ...identified in the central nervous system (CNS). The water channel aquaporin 1 (AQP1) is restricted to the apical domain of the choroid plexus epithelial cells. The AQP4 is abundantly expressed in astrocyte foot processes and ependymocytes facing capillaries and brain-cerebrospinal fluid (CSF) interfaces, whereas AQP9 is localized in tanycytes and astrocytes processes. The mRNA for other aquaporin homologs (i.e., AQP3, 5, and 8) have been recently found in cultured astrocytes. Based on their subcellular localization and data obtained from functional studies, it is assumed that aquaporins are implicated in water movements in nervous tissue and may play a role in central osmoreception, K+ siphoning, and cerebrospinal fluid formation. There have been recent reports describing different aquaporin-responses under pathologic states leading to brain edema. The data available provide a better understanding of the mechanisms responsible for brain edema and indicate that aquaporins are potential targets for drug development.
MicroRNAs in Cholangiopathies O’Hara, Steven P.; Gradilone, Sergio A.; Masyuk, Tetyana V. ...
Current pathobiology reports,
09/2014, Letnik:
2, Številka:
3
Journal Article
Odprti dostop
Cholangiocytes, the cells lining bile ducts, comprise a small fraction of the total cellular component of the liver, yet perform the essential role of bile modification and transport of biliary and ...blood constituents. Cholangiopathies are a diverse group of biliary disorders with the cholangiocyte as the target cell; the etiopathogenesis of most cholangiopathies remains obscure. MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. These small RNAs may not only be involved in the etiopathogenesis of disease, but are also showing promise as diagnostic and prognostic tools. In this brief review, we summarize recent work regarding the role of microRNAs in the etiopathogenesis of several cholangiopathies, and discuss their utility as prognostic and diagnostic tools.
Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3′,5′-cyclic monophosphate (cAMP)-induced translocation to the plasma ...membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%;
P < .001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%;
P < .05) and plasma (56%;
P < .05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL-induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3- and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional down-regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes. (H
epatology 2003;37:1026-1033.)