Huang, Bing

Abstract Background & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD‐causative genes, encoding for endoplasmic reticulum (ER)‐resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. Methods ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4‐phenylbutyric acid (4‐PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. Results The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4‐PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4‐PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. Conclusions Restoration of proteostasis in cystic cholangiocytes with 4‐PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

Abstract Abstract 4886 Detection of high molecular weight light chain oligomers in urinary exosomes of patients with AL amyloidosis. Background Exosomes are microvesicles that are part of the multivesicular body (MVB) pathway. They are created by the inward budding of the cell surface membrane and contain both surface bound membrane proteins and cytosolic proteins which can be used to identify the cell of origin. Immunoglobulin light chain amyloidosis (AL) occurs as the result of amyloid formation by the misfolding of monoclonal light chains (LC) and deposition of these amyloid fibrils in various soft tissues. This reaction requires the organization of the monoclonal LC's into protofibrils which are then weave into amyloid fibrils. This study was undertaken to determine whether urinary exosomes of glomerular origin contain intermediate species of amyloid formation. Method Urine samples from patients with AL, light chain deposition disease (LCDD), multiple myeloma (MM) and monoclonal clonal gammopathy of undetermined significance (MGUS) were collected. Urinary exosomes were isolated and separated into fractions by gradient centrifugation. Western blots were performed on the urinary exosome fractions using anti-kappa or anti-lambda antibodies. Glomerular fractions were identified using antibodies directed toward podocin. Results Urine samples were collected from 5 patients with AL, 2 from LCDD, 1 from MM and 1 MGUS. On the Western blot, immunoglobulin LC were seen in all exosomal fractions in patients with AL amyloidosis, LCDD, MM but not MGUS which is similar to normal controls (not shown). In patients with AL, oligomeric species were found in the highest concentrations in fraction 4 and 5 (Figure 1). Fraction 4 and 5 were also stained for podocin, a glomerular protein (not shown). The highest molecular weight species was ∼250 kd which corresponds to a LC decamer. High molecular weight species were also identified in 1 of 2 LCDD patients corresponding to a tetramer. The band was identified in fraction 10 which had polycystin-1 expression suggesting a tubular origin. No high molecular weight LC species was found in patients with MM or MGUS. Conclusion Our study found high molecular weight LC species corresponding to the intermediates involved in protofibril formation in urinary exosomes of patients with AL. Smaller (tetramer) high molecular weight LC species were also found in a patient with LCDD but not in patients with MM and MGUS. Not only were the high molecular weight LC species found exclusively in the diseases characterized by deposition of LC aggregates, they were also found in the segments of the nephron where the deposits were expected: glomerulus for AL and tubular epithelium for LCDD. This is consistent with our current understanding of the pathogenic mechanisms of these diseases. We believe urinary exosomes are a powerful tool in the study of diseases involving self-aggregation of monoclonal proteins. It has tremendous potential in both diagnostic and scientific research in this area. Disclosures Gertz: celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next‐generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three‐dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro , and the polycystic kidney disease–specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo . Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy–lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy‐related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP–protein kinase A–cAMP response element–binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A 1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three‐dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease–specific rat. Conclusion: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (H epatology 2018;67:1088–1108)

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein–coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m‐tolyl 5‐chloro‐2‐[ethylsulfonyl] pyrimidine‐4‐carboxylate [SBI‐115]), and (3) a combination of SBI‐115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA‐treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5−/− ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq) proteins was increased 2‐fold to 3‐fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5‐deficient TGR5−/−;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2‐fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI‐115 alone and by ∼50% when SBI‐115 was combined with pasireotide. Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197‐1218).

Abstract Primary cilia are multisensory organelles recently found to be absent in some tumor cells, but the mechanisms of deciliation and the role of cilia in tumor biology remain unclear. Cholangiocytes, the epithelial cells lining the biliary tree, normally express primary cilia and their interaction with bile components regulates multiple processes, including proliferation and transport. Using cholangiocarcinoma as a model, we found that primary cilia are reduced in cholangiocarcinoma by a mechanism involving histone deacetylase 6 (HDAC6). The experimental deciliation of normal cholangiocyte cells increased the proliferation rate and induced anchorage-independent growth. Furthermore, deciliation induced the activation of mitogen-activated protein kinase and Hedgehog signaling, two important pathways involved in cholangiocarcinoma development. We found that HDAC6 is overexpressed in cholangiocarcinoma and overexpression of HDAC6 in normal cholangiocytes induced deciliation and increased both proliferation and anchorage-independent growth. To evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by short hairpin RNA (shRNA) or by the pharmacologic inhibitor, tubastatin-A. Both approaches restored the expression of primary cilia in cholangiocarcinoma cell lines and decreased cell proliferation and anchorage-independent growth. The effects of tubastatin-A were abolished when cholangiocarcinoma cells were rendered unable to regenerate cilia by stable transfection of IFT88-shRNA. Finally, inhibition of HDAC6 by tubastatin-A also induced a significant decrease in tumor growth in a cholangiocarcinoma animal model. Our data support a key role for primary cilia in malignant transformation, provide a plausible mechanism for their involvement, and suggest that restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma. Cancer Res; 73(7); 2259–70. ©2013 AACR.

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. Cholangiocytes normally express primary cilia extending from their apical plasma membrane into the ductal lumen. Our data show that CCA cells are devoid of primary cilia both in vivo and in vitro; therefore, ciliary loss may be associated with cancer development. Histone deacetylase 6 (HDAC6) is localized to the cell cytoplasm and functions as a tubulin deacetylase, and the activation of HDAC6 promotes ciliary disassembly. Therefore, we hypothesized that in CCA, overexpression of HDAC6 induces the resorption of primary cilia leading to derepression of tumorigenic signaling pathways and development of a malignant phenotype. To test this hypothesis, we analyzed the effect of pharmacologic and molecular deciliation on the phenotype of two normal human cholangiocyte cell lines (H69, NHC) using chloral hydrate treatment or shRNA silencing of IFT88, respectively. We found that deciliation increased the proliferation rate of cholangiocytes and induced anchorage independent growth and invasion. Furthermore, deciliation induced the activation of MAPK and Hh signaling pathways by 6- and 3-fold, respectively. To begin to understand why CCA cells lack cilia, we assessed expression of HDAC6 by western blot analysis on two different CCA cell lines (Hucct-1 and KMCH); we found increased expression (by 100%) of HDAC6 compared to normal cultured cholangiocytes (H69). Interestingly, overexpression of HDAC6 correlated with the decreased amount of its target, acetylated α-tubulin. Using confocal immunofluorescence microscopy, we also observed that HDAC6 was overexpressed in liver specimens from patients with CCA compared to normals. Furthermore, the in vitro overexpression of HDAC6 in normal cells induced deciliation, increased proliferation, anchorage independent growth, and invasion. Finally, to evaluate the effect of cilia restoration on tumor cells, we targeted HDAC6 by shRNA silencing or by the inhibitor tubastatin-A, and found that HDAC6 downregulation restored the expression of primary cilia in a cancer cell line, decreased cell proliferation (by 30%), and anchorage-independent growth (by 50%). When tumor cells unable to regenerate cilia by the stably transfection of IFT88 shRNA were used, the effects of tubastatin-A were abolished. In summary, expression of primary cilia is decreased in CCA by a mechanism involving the overexpression of HDAC6. Experimental deciliation of normal cholangiocytes induces a malignant phenotype as shown by increased cell proliferation, anchorage independent growth, invasion, and activation of Hh and MAPK signaling pathways. We speculate that the restoration of primary cilia in tumor cells by HDAC6 targeting may be a potential therapeutic approach for cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-505. doi:1538-7445.AM2012-LB-505

Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic “saucer-shaped” morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca 2+ and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca 2+ -permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca 2+ in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo , by its specific agonist, 4αPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear. Cryptosporidium parvum , an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report that C. parvum infection of cholangiocytes recruits host-cell SGLT1, a Na + /glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampers C. parvum invasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion during C. parvum cellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general.