Proliferation and apoptosis
- Heterogeneity of the intrahepatic biliary epithelium

Cholangiocyte proliferation is coordinately regulated by a number of factors including gastrointestinal hormones/peptides, growth factors, cAMP and IP3/Ca2+/PKC pathways, nerves and bile acids[2,3,24,49,61,62,68,70-72,92-94]. Recent studies have shown that different sized cholangiocytes differentially proliferate or are damaged by apoptosis in response to injury, toxins, nerve resection and selected diets[2,26,48-52]. Following BDL, large but not small cholangiocytes proliferate with increases in basal and secretin-stimulated choleresis (Figure 3)[2,3,48]. We propose that large cholangiocytes selectively proliferate in response to BDL due to: (1) the predominant expression of VEGF in large compared to small cholangiocytes (Alpini et al, 2005, unpublished observation); and (2) the presence of the PBP mainly around large bile ducts, and less discernable around small bile ducts[44]. In support of this concept, in rats with BDL proliferation of the peribiliary plexus occurs only around large ducts[44]. Furthermore, we have recently demonstrated[94] that neutralization of VEGF levels of large cholangiocytes (by administration of a neutralizing anti-VEGF antibodies) reduces cholangiocyte growth typical of BDL rats[6]. In support of the concept that PBP and VEGF play a role in the regulation of large cholangiocyte function, hepatic artery ligation in BDL rats is associated with: (1) the disappearance of the PBP; (2) increased apoptosis and impaired proliferation of large cholangiocytes; and (3) decreased cholangiocyte VEGF secretion[93]. The effects of hepatic artery ligation on PBP and large cholangiocyte function were prevented by chronic administration of r-VEGF-A that, by maintaining the integrity of the PBP and large cholangiocyte proliferation, prevents bile duct damage following ischemic injury[93].
A number of gastrointestinal hormones/peptides have been shown to regulate the differential proliferative response of small and large cholangiocytes. We have shown that cholangiocytes express a1, a2, b1 and b2 thyroid hormone receptors and that the chronic administration of the thyroid hormone agonist, 3, 3¢, 5 L-tri-iodothyronine to BDL rats reduces in vivo the proliferation of large cholangiocytes[95], the only cholangiocyte subpopulation proliferating in this model[48]. In addition, in BDL rats we have shown that somatostatin inhibits the growth of large cholangiocytes by a decrease in cAMP levels[48]. Furthermore, gastrin inhibits large cholangiocyte proliferation in BDL rats by Ca2+/PKC-dependent inhibition of cAMP levels[72]
We have demonstrated that ovariectomy in BDL female rats reduces the proliferation of large cholangiocytes and induces a decrease in the expression of a and b estrogen receptors[69]. We propose that estrogens play a role in the management of chronic cholestatic liver diseases.
Recent studies have shown that nerves regulate the differential proliferative response of intrahepatic ducts.  We have shown that the activation of serotonin 1 A and 1 B receptors in cholangiocytes leads to the inhibition of large cholangiocyte proliferation in BDL rats[67]. Serotonin inhibition of large cholangiocyte proliferation was associated with activation of the IP3/Ca2+/PKC signaling pathway and the consequent inhibition of the cAMP/PKA/Src/ERK 1/2 pathway[67]. Since cholangiocytes secrete serotonin, we propose that serotonin limits the growth of intrahepatic bile ducts in the course of chronic cholestasis by an autocrine mechanism. Similarly, we have shown that cholangiocytes secrete NGF and that NGF secretion increases in proliferating BDL cholangiocytes compared to normal cholangiocytes[24]. In vivo, immunoneutralization of NGF (with an anti-NGF antibody) decreased large cholangiocyte proliferation[24]. The data suggest that NGF regulates cholangiocyte proliferation by an autocrine mechanism.
We have demonstrated that sensory innervation via α-calcitonin gene related peptide (a-CGRP) plays a role in adaptive proliferative responses of large cholangiocytes during cholestasis following BDL[96]. Specifically, we have shown that small and large murine cholangiocytes express the CGRP receptor components (calcitonin like receptor or CLR, receptor component protein or RCP and receptor activity modifying protein or RAMP1)[96]. Large, but not small, cholangiocytes proliferate in response to a-CGRP, proliferation that was blocked by CGRP[8-37], a-CGRP receptor antagonist[97]. a-CGRP stimulation of large cholangiocyte proliferation was associated with increased cAMP levels and phosphorylation of PKA and p38[97]. We observed a decrease in the number of proliferating large cholangiocytes in BDL knock-out mice (lacking a-CGRP) compared to BDL wild-type mice[96].
The role of the second messenger, cAMP, in the regulation of hepatic cell proliferation has been demonstrated in a number of animal models that stimulate hepatocyte and cholangiocyte proliferation via cAMP dependent mechanisms[26,50-52,54,98,99]. Following partial hepatectomy, there is an increase in intracellular cAMP levels in regenerating hepatocytes[100] and cholangiocytes[52].  Activation of Gas coupled receptors leads to activation of adenylyl cyclase and increased cAMP levels, whereas activation of Gai coupled receptors results in inhibition of AC activity and lowered intracellular cAMP levels[101]. cAMP response elements mediating transcriptional activation in response to increased intracellular cAMP levels have been identified[102]. In support of these findings, we have shown that chronic administration of forskolin to normal rats increased cAMP levels and the proliferation of large but not small cholangiocytes compared to rats receiving saline[70]. In purified cholangiocytes, forskolin increased large (but not small) cholangiocyte proliferation[70], which was blocked by Rp-cAMPs (a PKA inhibitor)[74], PP2 (a Src inhibitor)[103] and PD98059 (a MEK inhibitor)[104]. The effects of forskolin on large cholangiocyte proliferation were associated with increased phosphorylation of PKA, Src Tyr 139 and ERK1/2[70].  Maintenance of cAMP levels by forskolin administration prevents the effects of vagotomy on large cholangiocyte apoptosis (activation) and proliferation (inhibition)[26].
The acute administration of CCl4 to normal and BDL rats induces decreased cAMP levels and loss of function of large cholangiocytes at d 2 and transient elevation of cAMP levels in small cholangiocytes[50,51]. In these models, small cholangiocytes de novo express secretin receptors, a key component of the biliary proliferative and secretory mechanisms, suggesting that intracellular cAMP plays a key role in the: (1) de novo expression of large cholangiocyte phenotypes by small cholangiocytes (to compensate for loss of large cholangiocyte function); and (2) perhaps the differentiation of small cholangiocytes towards a cholangiocyte subpopulation that has the capacity to secrete and proliferate by cAMP-dependent pathway[50,51].
Following partial hepatectomy, both small and large cholangiocytes proliferate and participate in the regeneration of the intrahepatic biliary epithelium[52]. A single gavage dose of CCl4 to normal and BDL rats induces damage of large, cAMP-responsive cholangiocytes, whereas small cholangiocytes (resistant to CCl4) de novo proliferate and secrete (by the activation of the secretin receptor and secretin-stimulated cAMP levels) to compensate for the damage and loss of functional activity of large cholangiocytes[50,51]. The differential resistance of small and large cholangiocytes to CCl4 is presumably due to the presence of cytochrome P4502E1 (the enzyme that converts CCl4 to its radicals)[105]in large but not small cholangiocytes[50,51].  Chronic administration of the toxin, ANIT, induces proliferation of both small and large cholangiocytes, proliferation that (in contrast to other models including BDL)[26] was associated with enhanced apoptosis[47]. We propose that following ANIT or CCl4 feeding, the proliferation of small cholangiocytes may be due to the presence of cholangiocyte apoptosis in these models[47,50,51]. We also propose that the lack of small cholangiocyte proliferation in BDL rats may be due to the absence of cholangiocyte apoptosis in this model[26]. Similar to what is observed following acute CCl4 administration[50], the differential responses of small and large cholangiocytes to liver injury/toxins may be due to differential expression of other enzymes/proteins in small and large cholangiocytes. In support of this concept, phase I or mixed-function oxygenase enzymes (e.g., microsomal cytochrome P-450, aminopyrine-N-demethylases, G-6-PO4, and NADPH cytochrome C reductase) and phase II or glutathione redox cycle enzymes (e.g., GSH-peroxidase, UDP-glucuronosyltransferase, and glutathione-S-transferase) drug-metabolizing enzymes are heterogeneously expressed by cholangiocytes[50,81,106]. Similarly, since small murine cholangiocytes express annexin-V[107] (that regulates cell apoptosis)[108], this finding may explain partly why small ducts are more resistant than large ducts to some hepatic injury/toxins[50, 51]. In support of this concept, recent studies have shown that bcl-2 (an anti-apoptotic protein)[109] is expressed by small bile ducts in normal human liver and human liver with cirrhosis and focal nodular hyperplasia[110], a finding that may also explain partly the greater resistance of small cholangiocytes to damage[3,50,51].
In vitro treatment of normal cholangiocytes with taurocholate and taurolithocholate increases the proliferation of large but not small cholangiocytes[90]. Chronic feeding of taurocholate and taurolithocholate to normal rats induces the de novo expression of ASBT and activation of proliferation of small cholangiocytes, which do not constitutively express ASBT and are mitotically quiescent, and increases the proliferation of large cholangiocytes[49]. Prolonged feeding of ursodeoxycholate and tauroursodeoxycholate to BDL rats reduces the growth of large cholangiocytes[62] that selectively proliferate in this hyperplastic model[48].  Furthermore, depletion of endogenous bile acids reduced large cholangiocyte proliferation compared with BDL rats[92]. Re-infusion of taurocholate to bile acid-depleted rats prevented the decrease in cholangiocyte proliferation that was maintained at levels similar to those of BDL rats[92].
Histamine, an aminergic neurotransmitter, regulates many pathophysiological functions. Four G-protein coupled histamine receptors (H1, H2, H3 and H4) exist[111].  While H1 histamine receptors act via Gaq mobilizing [Ca2+]i[112], activation of H2 histamine receptors is modulated by Gas proteins, coupled to adenylyl cyclase[113]. H3 and H4 histamine receptors couple to Gai/o proteins that inhibit adenylyl cyclase[114]. Based upon our preliminary data, we propose a model in which the overall outcome of histamine on cholangiocyte growth is represented by a balance between its stimulatory (by activation of H1 and H2 histamine receptors)[91,115] and its inhibitory (by activation of H3 and H4 histamine receptors)[116, 117] actions on small and large cholangiocyte proliferation.  Specifically, we have shown that small but not large mouse cholangiocytes: (1) express the H1 histamine receptors and the calcium-dependent CaMK I (but not II or IV) protein kinase; and (2) proliferate in response to H1 histamine receptor agonists, proliferation that was blocked by BAPTA/AM, Gö6976 and W-7, a CAMK inhibitor[118]. IP3 (but not cAMP) levels were increased in small cholangiocytes treated with HTMT dimaleate. Chronic administration of the specific H3/H4R agonist (RAMH) to BDL rats decreased large cholangiocyte proliferation and cAMP levels compared to BDL rats treated with NaCl[116,117]. This inhibition is mediated through negative regulation of the cAMP-dependent PKA/ERK1/2 pathway[116,117].
The mechanisms by which different sized ducts proliferate or are damaged in response to various liver injury/toxins (e.g., BDL, partial hepatectomy, vagotomy, feeding of ANIT, bile acids or CCl4)[3,26,47-52] are unclear.  Furthermore, the pathophysiology of small cholangiocytes is undefined in these models. Based upon preliminary data and unpublished observations from our laboratory, we propose that neural/hormonal-dependent (cholinergic and adrenergic) activation of the Ca2+-dependent NFAT (Nuclear Factor of Activated T-lymphocytes) stimulates the proliferative response of small cholangiocytes, whereas neural/hormonal-dependent activation of the cAMP-dependent CREB stimulates the proliferation of large cholangiocytes. NFAT is a ubiquitous transcription factor that was initially described in T-lymphocytes.  Five isoforms of NFAT have been identified. Four of these isoforms (NFATc1 to c4) are regulated by Ca2+ signaling[119]. Preliminary data shows that Ca2+-dependent activation of NFATc1/c4 stimulates the proliferation of small cholangiocytes after CCl4-induced damage of cAMP-responsive large bile ducts[120]. Specifically, we have shown that small but not large normal rat cholangiocytes express the NFAT isoforms, NFAT c1 and c4[120]. CCl4 both in vivo and in vitro increased small cholangiocyte proliferation that was blocked by BAPTA/AM and 11R-VIVIT (NFAT inhibitor peptide)[121]. Furthermore, unpublished data from our laboratory show that the de novo growth of small cholangiocytes is regulated via adrenergic stimulation of Ca2+-dependent activation of NFATc1/c4 (Ca2+/calcineurin) and Sp1 (Ca2+/PKC). NFAT and Sp1 cooperatively interact to regulate proliferative phenotypes in other cell types[122].
Recent studies have shown that bile acids have cyto-protective effects against apoptosis in large cholangiocytes. Feeding of taurocholate to BDL rats (treated with a single dose of CCl4) prevents CCl4-induced damage of large cholangiocytes, whereas small cholangiocytes (which are de novo activated following CCl4-induced damage of large ducts)[50,51] remained mitotically dormant and unresponsive to secretin (Figure 3)[123]. In vitro, taurocholate prevented the inhibitory effects of CCl4 on apoptotic, proliferative and secretory capacity of large BDL cholangiocytes[123]. The protective effects of taurocholate against CCl4-induced damage of large BDL cholangiocytes are due to the activation of PI3-K and AKT expression[123]. Furthermore, feeding of taurocholate to BDL + vagotomy rats prevented vagotomy activation of large cholangiocyte apoptosis and inhibition of large cholangiocyte growth[124], effects that were abolished by wortmannin, a PI3-K inhibitor[125]. Functional ASBT expression as well as phosphorylation of Akt were reduced by vagotomy but restored by taurocholate feeding[124]. Chronic feeding of taurocholate prevented the increase in cholangiocyte apoptosis and the damage of large cholangiocyte proliferation induced by adrenergic denervation by 6-OHDA administration[126]. Taurocholate effects are mediated by the PI3K pathway, since the simultaneous administration of wortmannin reverses such effects[126]. In addition, the feeding of ursodeoxycholate and tauroursodeoxycholate to BDL + vagotomy rats prevented the activation of apoptosis and the loss of proliferation of large cholangiocytes observed in this model[127]. In this study[127], the protective effects of these two bile acids were neutralized by the simultaneous administration of BAPTA/AM (an intracellular Ca2+ chelator)[72] or Gö6976 (a PKC inhibitor)[65]. Both ursodeoxycholate and tauroursodeoxycholate increased IP3 and Ca2+ levels, together with enhanced phosphorylation of PKC-a[127]. The data suggests that bile acids are important in modulating large cholangiocyte proliferation in denervated livers. 

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