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.

Effects of dietary fish oil and corn oil on rat mammary tissue.

The effects, on the maternal mammary gland, of diets containing similar lipid percentages but differing in composition of polyunsaturated fatty acids (PUFA) have been assessed in rats during pregnancy and lactation. For this purpose, tuna fish oil (an n-3-PUFA-enriched oil) and corn oil (an n-6-PUFA-enriched oil) were included in diets at ratios such that the caloric inputs were the same as that of the control diet. As expected, the maternal diet affected the tissue composition of dams. Unexpectedly, only the tuna fish oil diet had an effect on pup growth, being associated with the pups being underweight between the ages of 11 and 21 days. The maternal mammary gland of rats fed the tuna fish oil diet displayed two main modifications: the size of cytoplasmic lipid droplets was increased when compared with those in control rats and the mammary epithelium showed an unusual formation of multilayers of cells. These results show that the tuna fish oil diet, during pregnancy and lactation, exerts specific effects on mammary cells and on the formation of lipid droplets. They suggest that this maternal diet affects the functioning of the mammary tissue.

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 ß-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 ß-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 ß-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 ß-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and ß-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 ß-casein synthesis and is a main regulator of ADRP expression.

Oleate and linoleate stimulate degradation of ß-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 beta-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 beta-casein levels. We demonstrate that the action of unsaturated fatty acids on the level of beta-casein is post-translational and requires protein synthesis. We have also observed that proteasome inhibitors potentiate beta-casein degradation, indicating that proteasomal activity can destroy some cytosolic protein(s) involved in the process that negatively controls beta-casein levels. Finally, lysosome inhibitors block the effect of unsaturated fatty acids on the cellular level of beta-casein. Our data thus suggest that the degradation of beta-casein occurs via the microautophagic pathway.

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.

[Nuclear organization and expression of milk protein genes]. / Organisation nucléaire et expression des gènes des protéines du lait.

Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.

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.

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 Drickamer's 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 alpha 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.

[The double-play of PP17/TIP47]. / Le double-jeu de la protéine TIP47.

A missing link in the understanding of the mechanisms of transport of the mannose 6-phosphate receptors has recently been discovered, following the identification of the protein TIP47. In association with Rab9-GTP, this protein is responsible for the return of the receptors from the late endosomes back to the trans-Golgi network. Curiously, the same protein called PP17b, was described as a placental protein twenty years ago, and more recently, as a blood marker for human uterine cervical cancer. The sequence of PP17b/TIP47 displays not only a strong homology with those of adipophilin and the perilipins, two proteins known to be involved in the intracellular traffic of lipid droplets but also PP17b/TIP47 is associated with the later. How this ubiquitous protein could participate in processes as different as the mannose 6-phosphate receptors traffic and the formation and/or traffic of lipid droplets? A tentative hypothesis is put forward.

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-alpha plus actinomycin-D-induced apoptosis. HIP/PAP anti-apoptotic effects against TNF-alpha 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.

Structure of the rabbit alphas1- and beta-casein gene cluster, assignment to chromosome 15 and expression of the alphas1-casein gene in HC11 cells.

Several casein (CSN) genes (CSN1, 2, 10 and alphas2-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.