Dynamic involvement of ATG5 in cellular stress responses.

Autophagy maintains cell and tissue homeostasis through catabolic degradation. To better delineate the in vivo function for autophagy in adaptive responses to tissue injury, we examined the impact of compromised autophagy in mouse submandibular glands (SMGs) subjected to main excretory duct ligation. Blocking outflow from exocrine glands causes glandular atrophy by increased ductal pressure. Atg5(f/-);Aqp5-Cre mice with salivary acinar-specific knockout (KO) of autophagy essential gene Atg5 were generated. While duct ligation induced autophagy and the expression of inflammatory mediators, SMGs in Atg5(f/-);Aqp5-Cre mice, before ligation, already expressed higher levels of proinflammatory cytokine and Cdkn1a/p21 messages. Extended ligation period resulted in the caspase-3 activation and acinar cell death, which was delayed by Atg5 knockout. Moreover, expression of a set of senescence-associated secretory phenotype (SASP) factors was elevated in the post-ligated glands. Dysregulation of cell-cycle inhibitor CDKN1A/p21 and activation of senescence-associated ß-galactosidase were detected in the stressed SMG duct cells. These senescence markers peaked at day 3 after ligation and partially resolved by day 7 in post-ligated SMGs of wild-type (WT) mice, but not in KO mice. The role of autophagy-related 5 (ATG5)-dependent autophagy in regulating the tempo, duration and magnitude of cellular stress responses in vivo was corroborated by in vitro studies using MEFs lacking ATG5 or autophagy-related 7 (ATG7) and autophagy inhibitors. Collectively, our results highlight the role of ATG5 in the dynamic regulation of ligation-induced cellular senescence and apoptosis, and suggest the involvement of autophagy resolution in salivary repair.

Deletion of ATG5 shows a role of autophagy in salivary homeostatic control.

Autophagy is a catabolic pathway utilized to maintain a balance among the synthesis, degradation, and recycling of cellular components, thereby playing a role in cell growth, development, and homeostasis. Previous studies revealed that a conditional knockout of essential member(s) of autophagy in a variety of tissues causes changes in structure and function of these tissues. Acinar cell-specific expression of knocked-in Cre recombinase through control of aquaporin 5 (Aqp5) promoter/enhancer (Aqp5-Cre) allows us to specifically inactivate Atg5, a protein necessary for autophagy, in salivary acinar cells of Atg5(f/f);Aqp5-Cre mice. There was no difference in apoptotic or proliferation levels in salivary glands of Atg5/Cre mice from each genotype. However, H&E staining and electron microscopy studies revealed modestly enlarged acinar cells and accumulated secretory granules in salivary glands of Atg5(f/f);Aqp5-Cre mice. Salivary flow rates and amylase contents of Atg5/Cre mice indicated that acinar-specific inactivation of ATG5 did not alter carbachol-evoked saliva and amylase secretion. Conversely, autophagy intersected with salivary morphological and secretory manifestations induced by isoproterenol administration. These results identified a role for autophagy as a homeostasis control in salivary glands. Collectively, Atg5(f/f);Aqp5-Cre mice would be a useful tool to enhance our understanding of autophagy in adaptive responses following targeted head and neck radiation or Sjögren syndrome.

Hoxb1 controls effectors of sonic hedgehog and Mash1 signaling pathways.

The diverse neuronal subtypes in the adult central nervous system arise from progenitor cells specified by the combined actions of anteroposterior (AP) and dorsoventral (DV) signaling molecules in the neural tube. Analyses of the expression and targeted disruption of the homeobox gene Hoxb1 demonstrate that it is essential for patterning progenitor cells along the entire DV axis of rhombomere 4 (r4). Hoxb1 accomplishes this function by acting very early during hindbrain neurogenesis to specify effectors of the sonic hedgehog and Mash1 signaling pathways. In the absence of Hoxb1 function, multiple neurons normally specified within r4 are instead programmed for early cell death. The findings reported here provide evidence for a genetic cascade in which an AP-specified transcription factor, Hoxb1, controls the commitment and specification of neurons derived from both alar and basal plates of r4.

Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor that has been implicated in the regulation of cell-specific gene expression mainly in hepatocytes and adipocytes but also in several other terminally differentiated cells. It has been previously demonstrated that the C/EBPalpha protein is functionally indispensable, as inactivation of the C/EBPalpha gene by homologous recombination in mice results in the death of animals homozygous for the mutation shortly after birth (Wang, N., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112). Here we show that C/EBPalpha -1-mice have defects in the control of hepatic growth and lung development. The liver architecture is disturbed, with acinar formation, in a pattern suggestive of either regenerating liver or pseudoglandular hepatocellular carcinoma. Pulmonary histology shows hyperproliferation of type II pneumocytes and disturbed alveolar architecture. At the molecular level, accumulation of glycogen and lipids in the liver and adipose tissues is impaired, and the mutant animals are severely hypoglycemic. Levels of c-myc and c-jun RNA are specifically induced by several fold in the livers of the C/EBPalpha -/- animals, indicating an active proliferative stage. Furthermore, immunohistologic detection with an antibody to proliferating cell nuclear antigen/cyclin shows a 5-10 times higher frequency of positively stained hepatocytes in C/EBPalpha -/- liver. These results suggest a critical role for C/EBPalpha in vivo for the acquisition of terminally differentiated functions in liver including the maintenance of physiologic energy homeostasis.

Expression of the liver-enriched transcription factors C/EBP alpha, C/EBP beta, HNF-1, and HNF-4 in preneoplastic nodules and hepatocellular carcinoma in rat liver.

The expression patterns of the liver-enriched transcription factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta and hepatocyte nuclear factor (HNF)-1 and HNF-4 were studied in liver nodules and hepatocellular carcinomas from male rats treated according to the resistant hepatocyte (RH) model. C/EBP alpha expression was lower at the transcriptional, mRNA, and protein levels in persistent nodules than in the respective surrounding livers. Expression was further decreased in the tumors. Transcriptional downregulation of C/EBP alpha gene expression was observed already in very early nodules, isolated 3 wk after partial hepatectomy in the RH model. However, no detectable changes were observed in preneoplastic nodules in the transcription or in steady-state mRNA levels of C/EBP beta, HNF-1, and HNF-4. A slight decrease in C/EBP beta protein and a more pronounced attenuation of HNF-1 and HNF-4 levels was observed in nodules, being 67%, 37%, and 46% of the levels in the corresponding surrounding livers, respectively. In conclusion, differential regulation of several transcription factors that are associated with the maintenance of the differentiated state of the hepatocytes was observed in preneoplastic and neoplastic liver lesions. This could have an impact on the regulation of a wide array of genes during liver carcinogenesis. Furthermore, the attenuation of C/EBP alpha expression, regarded as a negative growth regulator, could contribute to the proliferative advantage of nodules during liver carcinogenesis.

Analysis of antibody specificity against the third variable region of the envelope glycoprotein gp120 of HIV-1 in plasma from HIV-1-positive individuals residing in Brazil.

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70%) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60%) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

Analysis of antibody specificity against the third variable region of the envelope glycoprotein gp120 of HIV-1 in plasma from HIV-1-positive individuals residing in Brazil

1. Antibody specificity for the principal neutralization domain (PND) of the human immunodeficiency virus type 1 (HIV-1) was studied in plasma from 122 HIV-1-infected individuals residing in Brazil. 2. Using 8 overlapping sequential pentadecapeptides corresponding to the third variable region (V3) of 5 different HIV-1 isolates in an enzyme-linked immunosorbent assay (ELISA), a preferential recognition of the peptides with amino acid sequences corresponding to the HIV-1 isolates IIIB and MN (maximal reactivities of 60-70) compared to the isolates SC, WMJ-2 or RF (maximal reactivities below 60) was observed. 3. A difference was observed in the overall reactivity pattern to HIV-1 SC peptides of plasma collected from individuals residing in the Brazilian states of Rio de Janeiro and Bahia. However, a statistically significant increased recognition by Bahian plasma was only observed for the HIV-1 SC C55 peptide. 4. The mean CD4/CD8 ratio of the group of plasma with an isolate-restricted recognition of peptides (0.522 +/- 0.074) was significantly lower than that of the total group of plasma (1.00 +/- 0.18).

Differential patterns of expression of three C/EBP isoforms, HNF-1, and HNF-4 after partial hepatectomy in rats.

Regenerating liver provides a system for studying the mechanisms controlling regulated proliferation of differentiated hepatocytes. A set of transcription factors termed hepatocyte nuclear factors (HNF-1, -3, -4) and CCAAT/enhancer binding protein (C/EBP) isoforms are known to regulate several genes predominantly expressed in the liver. To assess whether these factors might be involved in the hepatocyte proliferation program, we have studied the expression of the three C/EBP isoforms C/EBP alpha, C/EBP beta, and C/EBP delta and the two hepatocyte-enriched transcription factors, HNF-1 and HNF-4, in rat liver at various time points after partial hepatectomy and sham operations using transcriptional "run-on" assays and Northern blot and Western blot experiments. We report here that partial hepatectomy in rats leads to dramatic changes in the pattern of expression of some of these genes. The three C/EBP isoforms are differentially regulated in response to partial hepatectomy and are likely to play different roles in determining the proliferation/differentiation state of hepatocytes. In particular, C/EBP alpha expression is rapidly down-regulated, whereas C/EBP delta is induced. C/EBP beta expression is also increased, although an increase is also observed after sham operation. The drastic decrease in C/EBP alpha under these conditions of active DNA synthesis and rapid cell proliferation further supports the concept of a potential incompatibility between high C/EBP alpha protein levels and cell proliferation. The patterns of transcriptional rates of HNF-1 and HNF-4 during the different stages of the regenerative process are similar. However, HNF-1 steady-state mRNA and protein levels are significantly changed while HNF-4 remains virtually unaffected, indicating that post-transcriptional mechanisms are also involved in the regulation of HNF-1 gene expression.

High level activity of the mouse CCAAT/enhancer binding protein (C/EBP alpha) gene promoter involves autoregulation and several ubiquitous transcription factors.

The promoter region of the mouse CCAAT-Enhancer Binding Protein (C/EBP alpha) gene is capable of directing high levels of expression of reporter constructs in various cell lines, albeit even in cells that do not express their endogenous C/EBP alpha gene. To understand the molecular mechanisms underlying this ubiquitous expression, we have characterized the promoter region of the mouse C/EBP alpha gene by a variety of in vitro and in vivo methods. We show that three sites related in sequence to USF, BTE and C/EBP binding sites and present in promoter region -350/+3, are recognized by proteins from rat liver nuclear extracts. The sequence of the C/EBP alpha promoter that includes the USF binding site is also capable of forming stable complexes with purified Myc+Max heterodimers and mutation of this site drastically reduces transcription of C/EBP alpha promoter luciferase constructs both in liver and non liver cell lines. In addition, we identify three novel protein-binding sites two of which display similarity to NF-1 and a NF kappa B binding sites. The region located between nucleotides -197 and -178 forms several heat-stable complexes with liver nuclear proteins in vitro which are recognized mainly by antibodies specific for C/EBP alpha. Furthermore, transient expression of C/EBP alpha and to a lesser extent C/EBP beta expression vectors, results in transactivation of a cotransfected C/EBP alpha promoter-luciferase reporter construct. These experiments support the notion that the C/EBP alpha gene is regulated by C/EBP alpha but other C/EBP-related proteins may also be involved.

Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins.

Site-directed mutagenesis was used to study the biological significance of a disulphide bridge and two N-linked oligosaccharides in the CD4-binding region of the envelope glycoproteins of human immunodeficiency virus type 1. Mutagenesis was performed in a phage M13 system at sites corresponding to the cysteine residue (amino acid 402) and the asparagine residues (390 and 447) of the env gene. The mutated env gene was inserted into a recombinant vaccinia virus under the control of the vaccinia virus 7.5K promoter and the expression of mutated env proteins was analysed by SDS-PAGE, a conventional indirect immunofluorescence assay and by a fluorescence-activated cell sorter. Cysteine 402 was found to be essential for the specific cleavage of gp160 into gp120 and gp41, and for intracellular transport of the protein to the cell surface. CD4-binding and syncytium formation assays demonstrated that the disulphide bridge of cysteine 402 stabilized a conformation essential for receptor binding as well as syncytium formation by CD4+ cells. No altered biological activity compared to that of the wild-type proteins could be detected for the mutant proteins lacking the N-glycosylation sites. These data show that the two conserved glycans attached to asparagine residues 390 and 447 do not play any active role in the formation of the disulphide bridge involving cysteine 402 or in the maintenance of an active conformation of the protein, despite their location within the functionally important CD4-binding region.

Production and characterization of a fragment containing the HIV-gp120 binding region of CD4 using a bovine papilloma virus (BPV) vector.

We have used a bovine papilloma virus (BPV) based mammalian cell expression vector consisting of the complete BPV genome and a human cytomegalovirus transcription unit for the production of soluble CD4. Mouse C-127 cells were transfected with vector DNA together with a selectable G418 resistance plasmid. Surviving clones were selected for high production using a solid phase ELISA based on the immobilization of supernatant-derived CD4 onto nitrocellulose paper and subsequent detection with anti-CD4 antibodies. The expressed protein was shown to bind HIV-gp120 and efficiently block HIV-1 infection in vitro. The possibility to use the above system for rapid production of defined glycoprotein fragments harboring defined functional regions, for the further elucidation of the functional role of CD4 in antigen presentation and cell to cell contact, and for possible intervention during HIV infection is discussed.

Cystein 402 of HIV gp 120 is essential for CD4-binding and resistance of gp 120 to intracellular degradation.

A DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp 120 was excised from an SV40-based expression vector containing gp 160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys402 was substituted for a serine, and metabolically labelled with [3H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp 120/gp 160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp 160, whereas gp 120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80-105k, suggesting increased sensitivity of mutant env gene products to proteolysis after cleavage to gp 120. Wild type gp 120 and gp 160 bound to CD4, whereas neither gp 160 nor gp 120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys402, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability of env gene products and for (ii) the physical stability of gp 120.

Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector.

A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium. Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.