Micheline Noël

Role of different epithelial cell types in liver ontogenesis, regeneration and neoplasia

The adult liver is a multicellular organ comprised of hepatocytes and several non-parenchymal cell popula tions. Beside the three well characterized sinusoidal cell subpopulations (i.e. Kupffer cells, endothelial cells and fat-storing cells), there are the epithelial cells of the biliary ductal structures and the mesothelial cells of the Glisson capsula (11,30,33,46,63). The liver of the vertebrate embryo develops as a diverticulum from an endomesodermal rudiment of the foregut. In the rat, the liver emerges at embryonic day 10 (E10) and acquires clear anatomic limits around E12 (10,42,60,67). The parenchymal cells of El0-13 rat liver are morphologically homogenous, but are different from those at El5-17. Biliary ductal structures are identifiable at El 7-18. Endothelial cells and Kupffer cells are already present by E10. Hemopoietic cells persist in the liver beyond E12 and are excluded early after birth (55). Up to the weanling period, the hepatocytes represent a diploid epithelial population and exhibit an active but transient prolifera tive activity, and thereafter they undergo polyploidization (12,52). While the growth rate of hepatocytes throughout the normal adult life is extremely low, they are capable of rapid proliferation in response to insult or crisis (33,63). However, normal reconstruction of the tissue implies the growth of the other cell types as well. In fact, there is already substantial evidence suggesting wide differences in the dynamics of the various hepatic cell populations during regeneration in response to chemical intoxication. During chemical hepatocarcinogenesis the emergence of hepatocellular carcinomas in rats is a progressive cellular process characterized by the successive appearance of epithelial cell populations exhibiting distinct phenotypes (9,24,31,62,70,72). As a result of new experimental approaches, significant progress has been made during the last few years in the

Growth and functional activities of neonatal and adult rat hepatocytes cultured on fibronectin coated substratum in serum-free medium

SummaryHepatocytes isolated from neonatal (NN) and adult (AD) rats were seeded on fibronectin coated substratum and cultured in arginine-free medium supplemented with various combinations of insulin, dexamethasone, triiodothyronine (T3), albumin, and transferrin, in presence or absence of fibronectin depleted serum (FDS). The main finding is that in response to certain hormone mixtures, both NN and AD hepatocytes can be stimulated to proliferate, as revealed by an increase in cell number, a [3H]thymidine incorporation into nuclei, and extractable DNA as well as the appearance of mitotic figures. Moreover, this proliferative activity is associated with changes in hepatocyte ploidy. However, the proliferative response of NN hepatocytes to hormone action is much different from that of AD hepatocytes, and the addition of FDS amplifies this activity in NN but inhibits it in AD hepatocyte cultures. Measurements of tyrosine aminotransferase and lactate dehydrogenase activities indicate a good preservation of NN and AD hepatocyte functional integrity under certain culture conditions. A good maintenance of albumin production in NN and AD hepatocyte cultures requires the presence of dexamethasone, whereas theα-fetoprotein production in NN hepatocyte cultures is reduced quite rapidly under most conditions. Noα-fetoprotein is detectable in AD hepatocyte cultures.

Molecular characterization of a human cation-Cl- cotransporter (SLC12A8A, CCC9A) that promotes polyamine and amino acid transport.

Cation‐Cl− cotransporters (CCCs) belong to a large family of proteins that includes 9 isoforms, two of which have still not been ascribed a transport function (CCC8 and CCC9) while the others are all known to promote Cl−‐coupled Na+ and/or K+ movement at the cell surface. The CCCs are also included in a larger family termed amino acid‐polyamine‐organocation carriers (APCs). In contrast to the CCCs, however, polyamine (PA) transporters have thus far been isolated from unicellular species exclusively and do not all belong to the APC family. In this work, we have found that a splice variant of CCC9 (CCC9a) promotes PA‐amino acid transport at the surface of HEK‐293 cells. We have also found that the influx of PAs in CCC9a‐expressing cells is inhibited by pentamidine as well as furosemide, and that it increases further in the presence of specific amino acids but not of Na+, K+, or Cl−. Hence, a group of substrates that are directly transported by CCC9 and the molecular identity of a PA transport system in animal cells may have been uncovered for the first time. These findings are of special interest given that intracellular PAs play a key role in cell proliferation. J. Cell. Physiol. 220: 680–689, 2009.

Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants

The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.

Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.

Aquaporins Mediate Silicon Transport in Humans

In animals, silicon is an abundant and differentially distributed trace element that is believed to play important biological functions. One would thus expect silicon concentrations in body fluids to be regulated by silicon transporters at the surface of many cell types. Curiously, however, and even though they exist in plants and algae, no such transporters have been identified to date in vertebrates. Here, we show for the first time that the human aquaglyceroporins, i.e., AQP3, AQP7, AQP9 and AQP10 can act as silicon transporters in both Xenopus laevis oocytes and HEK-293 cells. In particular, heterologously expressed AQP7, AQP9 and AQP10 are all able to induce robust, saturable, phloretin-sensitive silicon transport activity in the range that was observed for low silicon rice 1 (lsi1), a silicon transporter in plant. Furthermore, we show that the aquaglyceroporins appear as relevant silicon permeation pathways in both mice and humans based on 1) the kinetics of substrate transport, 2) their presence in tissues where silicon is presumed to play key roles and 3) their transcriptional responses to changes in dietary silicon. Taken together, our data provide new evidence that silicon is a potentially important biological element in animals and that its body distribution is regulated. They should open up original areas of investigations aimed at deciphering the true physiological role of silicon in vertebrates.

Phosphoregulation of K+ -Cl− cotransporters during cell swelling: Novel insights

The K+‐Cl− cotransporters (KCCs) belong to the cation‐Cl− cotransporter family and consist of four isoforms and many splice variants. Their main role is to promote electroneutral efflux of K+ and Cl− ions across the surface of many cell types and, thereby, to regulate intracellular ion concentration, cell volume, and epithelial salt movement. These transport systems are induced by an increase in cell volume and are less active at lower intracellular [Cl−] (Cli), but the mechanisms at play are still ill‐defined. In this work, we have exploited the Xenopus laevis expression system to study the role of lysine‐deficient protein kinases (WNKs), protein phosphatases 1 (PP1s), and SPS1‐related proline/alanine‐rich kinase (SPAK) in KCC4 regulation during cell swelling. We have found that WNK4 and PP1 regulate KCC4 activity as part of a common signaling module, but that they do not exert their effects through SPAK or carrier dephosphorylation. We have also found that the phosphatases at play include PP1α and PP1γ1, but that WNK4 acts directly on the PP1s instead of the opposite. Unexpectedly, however, both cell swelling and a T926A substitution in the C‐terminus of full‐length KCC4 led to higher levels of heterologous K+‐Cl− cotransport and overall carrier phosphorylation. These results imply that the response to cell swelling must also involve allosteric‐sensitive kinase‐dependent phosphoacceptor sites in KCC4. They are thus partially inconsistent with previous models of KCC regulation.

Identification of key residues involved in Si transport by the aquaglyceroporins

For the aquaporins, selectivity to water is ensured to a large extent by an arginine filter within the pore of the channel. Carpentier et al. find that selectivity to silicon is also controlled by this filter but with the involvement of additional residues that are within or close to the pore.

Progenitor Cells in Embryonic and Post-Natal Rat Livers, Their Growth and Differentiation Potential

The adult liver is a multicellular organ composed of hepatocytes, biliary epithelial cells, mesothelial cells and several types of non-epithelial cells. The organ exhibits many metabolic fonctions, the main ones being the production of bile and plasma proteins (Daoust and Brauer, 1958). Liver damage is frequent in humans, mostly from genetic alterations through inheritance or acute metabolic failures as a result of exposure to toxic chemicals (Bucher and McGowan, 1979). The most dramatic consequence of liver damage is liver failure (Martin and Feldmann, 1983) which requires organ transplantation, a procedure that has many obvious drawbacks (Keeffe, 1991). An alternative approach would be to transplant normal liver cells that have the potential of re-populating the organ and exerting the specialyzed functions of the mature hepatic tissue. This cell transfer approach has already been used with good success for other tissues, such as the hemopoietic tissue cell system(Thomas et al., 1992; Carlo-Stella et al., 1992), where rare stem cells have the capacity to grow massively and to differentiate along the various hemopoietic cell lineages.

Ablation of Potassium-Chloride Cotransporter Type 3 (Kcc3) in Mouse Causes Multiple Cardiovascular Defects and Isosmotic Polyuria

Inactivation of Kcc3 in a mixed 129/Sv×C57BL/6 mouse background has been previously found to increase systemic blood pressure (BP) through presumed neurogenic mechanisms. Yet, while this background is generally not considered ideal to investigate the cardiovascular system, KCC3 is also expressed in the arterial wall and proximal nephron. In the current study, the effects of Kcc3 ablation was investigated in a pure rather than mixed C57BL/6J background under regular- and high-salt diets to determine whether they could be mediated through vasculogenic and nephrogenic mechanisms. Aortas were also assessed for reactivity to pharmacological agents while isolated from the influence of sympathetic ganglia. This approach led to the identification of unforeseen abnormalities such as lower pulse pressure, heart rate, aortic reactivity and aortic wall thickness, but higher diastolic BP, left ventricular mass and urinary output in the absence of increased catecholamine levels. Salt loading also led systolic BP to be higher, but to no further changes in hemodynamic parameters. Importantly, aortic vascular smooth muscle cells and cardiomyocytes were both found to express KCC3 abundantly in heterozygous mice. Hence, Kcc3 inactivation in our model caused systemic vascular resistance and ventricular mass to increase while preventing extracellular fluid volume to accumulate. Given that it also affected the physiological properties of aortas in vitro, vasculogenic mechanisms could therefore account for a number of the hemodynamic abnormalities observed.