A targeted deletion/insertion in the mouse Pcsk1 locus is associated with homozygous embryo preimplantation lethality, mutant allele preferential transmission and heterozygous female susceptibility to dietary fat.

Proprotein convertase 1 (PC1) is a neuroendocrine proteinase involved in the proteolytic activation of precursors to hormones and neuropeptides. To determine the physiological importance of PC1, we produced a mutant mouse from embryonic stem cells in which its locus (Pcsk1) had been inactivated by homologous recombination. The inactivating mutation consisted of a 32.7-kb internal deletion and a 1.8 kb insertion of the bacterial neomycin resistance gene (neo) under the mouse phosphoglycerate kinase 1 protein (PGKneo). Intercross of Pcsk1(+/-) mice produced no Pcsk1(-/-) offspring or blastocysts; in addition, more than 80% of the offspring were Pcsk1(+/-). These observations suggested that the mutation caused preimplantation lethality of homozygous embryos and preferential transmission of the mutant allele. Interestingly, RT-PCR analysis on RNA from endocrine tissues from Pcsk1(+/-) mice revealed the presence of aberrant transcripts specifying the N-terminal half of the PC1 propeptide fused to neo gene product. Mass spectrometric profiles of proopiomelanocortin-derived peptides in the anterior pituitary were similar between Pcsk1(+/-) and Pcsk1(+/+) mice, but significantly different between male and female mice of the same genotype. Relative to their wild-type counterparts, female mutant mice exhibited stunted growth under a low fat diet, and catch-up growth under a high-fat diet. The complex phenotype exhibited by this Pcsk1 mutant mouse model may be due to PC1 deficiency aggravated by expression of aberrant gene products from the mutant allele.

Increased stress-induced analgesia in mice lacking the proneuropeptide convertase PC2.

Many neuropeptides involved in pain perception are generated by endoproteolytic cleavages of their precursor proteins by the proprotein convertases PC1 and PC2. To investigate the role of PC2 in nociception and analgesia, we tested wild-type and PC2-null mice for their responses to mechanical and thermal nociceptive stimuli, before and after a short swim in cold or warm water. Basal responses and responses after a cold swim were similar between the two groups. However, after a short forced swim in warm water, PC2-null mice were significantly less responsive to the stimuli than wild-type mice, an indication of increased opioid-mediated stress-induced analgesia. The enhanced analgesia in PC2-null mice may be caused by an accumulation of opioid precursor processing intermediates with potent analgesic effects, or by loss of anti-opioid peptides.

Expression and transient nuclear translocation of proprotein convertase 1 (PC1) during mouse preimplantation embryonic development.

Preimplantation embryos express a number of hormones, neuropeptides, and membrane receptors known to derive from proteolytic activation of their precursors by the seven-member family of subtilisin-like, calcium-dependent serine proteinases known as proprotein convertases (PCs). The goal of this study was to determine the pattern of PC expression in mouse preimplantation embryos. Transcripts for all PCs, except PC2, were detected by reverse transcription-polymerase chain reaction (RT-PCR) in unfertilized and fertilized eggs. Furin, PACE4, PC1, and PC7 transcripts remained present at subsequent stages of preimplantation embryonic development, whereas the levels of transcripts for PC4 and PC5 gradually disappeared after the 2-cell stage. Proprotein convertase 1 (PC1) expression was further examined at the protein level. Immunoblotting revealed the presence of the zymogen and mature forms of this enzyme in eggs and embryos. Immunofluorescence laser confocal microscopy showed PC1-specific staining throughout the cytoplasm of unfertilized eggs. After fertilization, surprisingly, the staining was concentrated in pronuclei. It relocated to the cytoplasm at postzygotic stages and was particularly strong at junctions between blastomeres. The nuclear translocation of PC1 in fertilized eggs is probably mediated by its prodomain. Indeed, when transduced in human colon carcinoma LoVo cells, a mutant proPC1 incapable of cleaving off its prodomain was shown to accumulate in the nucleus. Furthermore, when N-terminally fused to green fluorescent protein, this domain was able to direct the reporter protein to the nucleus of these cells. Collectively, these data establish that eggs and preimplantation embryos express various PCs necessary for proteolytic activation of precursors of hormones and growth factors. They also raise the possibility of a nuclear function for PC1 during zygote formation.

Altered processing of the neurotensin/neuromedin N precursor in PC2 knock down mice: a biochemical and immunohistochemical study.

Neurotensin (NT) and neuromedin N (NN) are generated by endoproteolytic cleavage of a common precursor molecule, pro-NT/NN. To gain insight into the role of prohormone convertases PC1, PC2, and PC7 in this process, we investigated the maturation of pro-NT/NN in the brain of PC7 (PC7-/-), PC2 (PC2-/-), and/or PC1 (PC1+/- and PC2-/-; PC1+/-) knock down mice. Inactivation of the PC7 gene was without effect, suggesting that this convertase is not involved in the processing of pro-NT/NN. By contrast, there was a 15% decrease in NT and a 50% decrease in NN levels, as measured by radioimmunoassay, in whole brain extracts from PC2 null as compared with wild type mice. Using immunohistochemistry, we found that this decrease in pro-NT/NN maturation products was uneven and that it was most pronounced in the medial preoptic area, lateral hypothalamus, and paraventricular hypothalamic nuclei. These results suggest that PC2 plays a critical role in the processing of pro-NT/NN in mouse brain and that its deficiency may be compensated to a regionally variable extent by other convertases. Previous data have suggested that PC1 might be subserving this role. However, there was no change in the maturation of pro-NT/NN in the brain of mice in which the PC1 gene had been partially inactivated, implying that complete PC1 knock down may be required for loss of function.

Involvement of matrix metalloproteinases in the adipose conversion of 3T3-L1 preadipocytes.

When mouse 3T3-L1 preadipocytes are induced to differentiate into adipocytes, they change from an extended fibroblast-like morphology to a rounded one. This change most likely occurs through extracellular matrix remodelling, a process known to be mediated in part by matrix metalloproteinases (MMPs). In this study, we have shown by semi-quantitative reverse transcriptase-PCR, zymographic and immunoblot analysis that MMP-2, MMP-9 and membrane type 1 (MT1)-MMP are regulated during adipose conversion. To assess the importance of MMPs for adipocytic differentiation we have used MMP-specific inhibitors as well as neutralizing antibodies. Treatment of 3T3-L1 preadipocytes with the broad MMP inhibitor Ilomastat or the more restricted MMP-2 Inhibitor I prevented their differentiation into adipocytes in a dose-dependent manner, as evidenced by absence of triglyceride accumulation. Inhibitor treatment prevented the fibronectin-network degradation, as well as the induction of the genes for peroxisome-proliferator-activated receptor gamma and adipsin, two adipocyte phenotype markers. Inhibitor treatment was effective when applied during the early stages of adipocytic conversion, whereas inhibitor treatment during later stages had little effect. Inhibitor treatment did not inhibit clonal mitotic expansion; nor did it affect the expression pattern of the adipogenic transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) or its nuclear translocation. It did, however, markedly reduce C/EBPbeta DNA-binding capacity. Taken together, these results suggest that MMPs, and notably MMP-2 and MMP-9, may be necessary mediators of adipocytic differentiation of 3T3-L1 cells.

Proprotein convertases are important mediators of the adipocyte differentiation of mouse 3T3-L1 cells.

Mouse 3T3-L1 cells are widely used to study adipocyte differentiation in vitro. When treated with insulin, dexamethasone and isobutylmethylxanthine these fibroblastic cells differentiate into round triglyceride-rich adipocytes. Because several proteins implicated in adipocyte differentiation (e.g. type 1 IGF receptors) are proteolytically activated by endoproteinases of the proprotein convertase family, we sought to determine whether these endoproteinases are crucial for adipose conversion. In this study, we show that expression of the proprotein convertases PACE4, PC7 and furin increases when 3T3-L1 cells are induced to differentiate into adipocytes. The differentiation was blocked in transfected cells expressing alpha1-antitrypsin Portland or in normal cells pre-treated with the synthetic inhibitor decanoyl-RVKR-chloromethylketone. Both inhibitors are known to specifically inactivate proprotein convertases. The block was associated with impaired proteolytic activation of proIGF-1 receptor, absence of induction of the adipogenic transcriptional factor PPARgamma and marked reduction of the nuclear translocation of the C/EBPbeta factor. Taken together, these data constitute evidence that proprotein convertases are crucial mediators of adipogenesis.