Alain Pauloin

HIP/PAP stimulates liver regeneration after partial hepatectomy and combines mitogenic and anti-apoptotic functions through the PKA signaling pathway

The HIP/PAP (=human Reg‐2) C‐type lectin encoding gene is activated in primary liver cancers. In normal liver, the protein is undetectable in normal mature hepatocytes and found only in some ductular cells, representing potential hepatic progenitor cells. The aim of this study was to examine the consequences of human HIP/PAP expression in the liver of transgenic mice. We demonstrated that HIP/PAP stimulated liver regeneration after partial hepatectomy. To further investigate the enhanced liver regeneration observed in vivo, primary cultures of hepatocytes were used to evaluate the mitogenic and anti‐apoptotic properties of HIP/PAP. HIP/PAP increased hepatocyte DNA synthesis and protected hepatocytes against TNF‐α plus actinomycin‐D‐induced apoptosis. HIP/PAP anti‐apoptotic effects against TNF‐α were clearly demonstrated when protein kinase A activity was specifically inhibited by KT5720, and HIP/PAP stimulated PKA‐dependent phosphorylation of the proapoptotic Bad protein at Ser‐112, suggesting that HIP/PAP may compete with cAMP to stimulate PKA activity. Overall, our results led us to propose a new role for a C‐type lectin, HIP/PAP, as a hepatic cytokine that combines mitogenic and anti‐apoptotic functions regarding hepatocytes, and consequently acts as a growth factor in vivo to enhance liver regeneration.—Simon, M.‐T., Pauloin, A., Normand, G., Lieu, H.‐T., Mouly, H., Pivert, G., Carnot, F., Tralhao, J. G., Bréchot, C., Christa, L. HIP/PAP stimulates liver regeneration after partial hepatectomy and combines mitogenic and antiapoptotic functions through the PKA signaling pathway. FASEB J. 17, 1441–1450 (2003)

HIP/PAP accelerates liver regeneration and protects against acetaminophen injury in mice†

Human hepatocarcinoma‐intestine‐pancreas/pancreatic‐associated protein HIP/PAP is a secreted C‐type lectin belonging to group VII, according to Drickamers classification. HIP/PAP is overexpressed in liver carcinoma; however, its functional role remains unclear. In this study, we demonstrate that HIP/PAP is a paracrine hepatic growth factor promoting both proliferation and viability of liver cells in vivo. First, a low number of implanted hepatocytes deriving from HIP/PAP‐transgenic mice (<1:1,000) was sufficient to stimulate overall recipient severe combined immunodeficiency liver regeneration after partial hepatectomy. After a single injection of HIP/PAP protein, the percentages of bromodeoxyuridine‐positive nuclei and mitosis were statistically higher than after saline injection, indicating that HIP/PAP acts as a paracrine mitogenic growth factor for the liver. Comparison of the early events posthepatectomy in control and transgenic mice indicated that HIP/PAP accelerates the accumulation/degradation of nuclear phospho–signal transducer activator transcription factor 3 and tumor necrosis factor α level, thus reflecting that HIP/PAP accelerates liver regeneration. Second, we showed that 80% of the HIP/PAP‐transgenic mice versus 25% of the control mice were protected against lethal acetaminophen‐induced fulminate hepatitis. A single injection of recombinant HIP/PAP induced a similar cytoprotective effect, demonstrating the antiapoptotic effect of HIP/PAP. Comparison of Cu/Zn superoxide dismutase activity and glutathione reductase‐like effects in control and transgenic liver mice indicated that HIP/PAP exerts an antioxidant activity and prevents reactive oxygen species‐induced mitochondrial damage by acetaminophen overdose. In conclusion, the present data offer new insights into the biological functions of C‐type lectins. In addition, HIP/PAP is a promising candidate for the prevention and treatment of liver failure. (HEPATOLOGY 2005;42:618–626.)

Roads taken by milk proteins in mammary epithelial cells

Abstract Mammary cell secretory pathways are now well known: milk proteins initially appear over the endoplasmic reticulum, transiently associate with elements of the Golgi complex, then concentrate in post-Golgi secretory vesicles where caseins are detectable in aggregated form (casein micelles). Mechanisms controlling the transport of milk proteins between these different organelles are less well known. Study of transport of caseins in mammary cells from goats naturally deficient in αs1-casein has made it possible to show that quality control is associated with the forward transport of caseins out of the endoplasmic reticulum. Transport of caseins between endoplasmic reticulum, Golgi saccules, trans Golgi network (TGN) and secretory vesicles is under the control of protein kinase A and phospholipase D. Last steps of exocytosis of milk proteins are stimulated by prolactin-induced arachidonic acid release suggesting a role of PLA2. The mammary epithelial cell also internalises plasma-borne proteins (hormones, growth factors, transferrin, immunoglobulins) in part via clathrin-coated vesicles, and carries many of them, to milk, by transcytosis. As shown in rodents and rabbits, sorting occurs, for the different ligands internalised, in endosomes. Transferrin is mainly recycled to the basal membrane, markers of the basal membrane are rapidly carried to the Golgi region and prolactin is transcytosed across late endosomes and secretory vesicles. Visualisation of the intracellular pathway of prolactin, in mammospheres from lactating rabbits, reveals that transcytosis occurs via a tubulo-vesicular network across the cell and that the roads of endocytosis and exocytosis are interconnected.

Oleate and linoleate stimulate degradation of β-casein in prolactin-treated HC11 mouse mammary epithelial cells.

Although virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We have used the mammary epithelial cell line HC11 to evaluate the potential connections between the lipid and protein synthetic pathways. We show that unsaturated fatty acids induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of adipose differentiation-related protein. Unexpectedly, the cellular level of β-casein, accumulated under lactogenic hormone treatment, decreases following treatment of the cells with unsaturated fatty acids. In contrast, saturated fatty acids have no significant effect on either cytoplasmic lipid droplet proliferation or cellular β-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of β-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate β-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls β-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of β-casein. Our data thus suggest that the degradation of β-casein occurs via the microautophagic pathway.

Prolactin and epidermal growth factor stimulate adipophilin synthesis in HC11 mouse mammary epithelial cells via the PI3-kinase/Akt/mTOR pathway

The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of β-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose-response inhibition of PRL-induced β-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and β-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in β-casein synthesis and is a main regulator of ADRP expression.

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.

Structure of the rabbit αs1- and β-casein gene cluster, assignment to chromosome 15 and expression of the αs1-casein gene in HC11 cells

Abstract Several casein ( CSN ) genes ( CSN1 , 2 , 10 and αs2-CSN ) have been described and shown to be clustered in mouse, man and cattle. These genes are expressed simultaneously in the mammary gland during lactation, but they are silent in most mammary cell lines, even in the presence of lactogenic hormones. However, it has been shown that the CSN2 gene, and this gene only, can be induced in certain mammary cell lines, such as HC11. In the present paper, we describe three overlapping bacterial artificial chromosome (BAC) clones which harbor both the rabbit CSN1 and CSN2 genes. These two genes are in a convergent orientation, separated by an intergenic region of 15 kb. DNA from one of the CSN /BAC clones was used as a probe for in situ hybridization to show that the CSN1 and CSN2 gene cluster is located on chromosome 15 band q23 and not on chromosome 12 as had been previously reported. Each of the three CSN /BAC DNAs was transfected into HC11 cells. In the presence of lactogenic hormones, the rabbit CSN1 gene was clearly expressed from all three CSN /BAC DNAs, whereas the rabbit CSN2 gene, which at the most possesses a 1 kb upstream region in one of the CSN /BAC DNAs, was not expressed at detectable levels on Northern blots. The transfected HC11 cells now express both rabbit CSN1 and mouse CSN2 genes. These transfected cells will be used as a model to study the role of CSN1 in milk protein secretion.

Purification and Characterization of Two Casein Kinase Type II Isozymes from Bovine Brain Gray Matter

Abstract: Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono‐Q steps. The phosphocellulose eluate showed two BBGM‐CK II activities. The first minor component (BBGM‐CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM‐CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., α (40 kDa), α′ (38 kDa), and β (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM‐CK IIa and b showed chromatographic and biochemical differences including differing Km for ATP and GTP and Ki for heparin and 2,3‐bisphosphoglycerate. The properties of the main peak (BBGM‐CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM‐CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM‐CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin β‐light chain. The unusual chromatographic behavior and biochemical properties of these BBGM‐CK II isozymes, compared with the classical CK II, could be explained at least in part by their subunit ratios.

Interactions between the rabbit CSN1 gene and the nuclear matrix of stably transfected HC11 mammary epithelial cells vary with its level of expression.

The expression of casein genes is specific to the mammary gland and maximal during lactation. However, among the numerous mammary cell lines described so far, only a few express some casein genes. The regulatory regions of casein genes have been largely described but the mechanisms explaining the mammary specific expression of these genes, and their silencing in most mammary cell lines, have not yet been fully elucidated. To test the hypothesis that the nuclear location of the casein genes may affect their expression, we transfected HC11 mouse mammary cell line with a 100 kb DNA fragment surrounding the rabbit alpha S1 casein gene. We derived stable clones which express or not the transfected rabbit casein gene, in the same cellular context, independently of the number of transgene copies. Metaphase spreads were prepared from the different clones and the transfected genes were localized. Unexpectedly, we observed that in the original HC11 cell line the number of chromosomes per metaphase spread is close to 80, suggesting that HC11 cells have undergone a duplication event, since the mouse karyotype is 2n = 40. In alpha S1 casein expressing cells, the expression level does not clearly correlate with a localization of the transfected DNA proximal to the centromeres or the telomeres. Analysis of the localization of the transfected DNA in nuclear halos allows us to conclude that when expressed, transfected DNA is more closely linked to the nuclear matrix. The next step will be to study the attachment of the endogenous casein gene in mammary nuclei during lactation.

The perilipin‐2 (adipophilin) coat of cytosolic lipid droplets is regulated by an Arf1‐dependent mechanism in HC11 mouse mammary epithelial cells

The cytosolic lipid droplets (cLDs) store excess intracellular lipids, and perilipin‐2 is believed to protect cLDs from degradation. Here, we investigated the role of the small G‐protein Arf1 and the proteasome in the fates of perilipin‐2 and cLDs. In oleate‐loaded cells, upon brefeldin A (BFA) treatment, perilipin‐2 remained associated with cLDs for at least 30 min before significant release, and proteasomal degradation‐mediated decrease was observed. Interestingly, the cLD population did not mimic the decline in perilipin‐2. We tested several chemical modulators of regulators of Arf1 activity on the association of perilipin‐2 with cLDs. QS11 and Exo2 accelerated the reduction in perilipin‐2, although less than BFA. In contrast, Exo1 unexpectedly slowed down its degradation. Correlatively, BFA, QS11, and Exo2 enhanced the dissociation of perilipin‐2 from cLDs, whereas Exo1 inhibited it. There was a synergistic effect of BFA with Exo2 and QS11, and of Exo2 with QS11, whereas Exo1 antagonized the effect of BFA without affecting that of Exo2 or QS11. We concluded that the Arf1 complex regulates the association of perilipin‐2 with cLDs. Additionally, MG132 and BFA modified the number of cLDs over a relatively short period.