Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization.

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice.

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.

Disruption of Stat3 reveals a critical role in both the initiation and the promotion stages of epithelial carcinogenesis.

Constitutive activation of signal transducer and activator of transcription 3 (Stat3) has been found in a wide spectrum of human malignancies. Here, we have assessed the effect of Stat3 deficiency on skin tumor development using the 2-stage chemical carcinogenesis model. The epidermis of Stat3-deficient mice showed a significantly reduced proliferative response following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) because of a defect in G1-to-S-phase cell cycle progression. Treatment with the tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA) resulted in a significant increase in the number of keratinocyte stem cells undergoing apoptosis in the bulge region of hair follicles of Stat3-deficient mice compared with nontransgenic littermates. Notably, Stat3-deficient mice were completely resistant to skin tumor development when DMBA was used as the initiator and TPA as the promoter. Abrogation of Stat3 function using a decoy oligonucleotide inhibited the growth of initiated keratinocytes possessing an activated Ha-ras gene, both in vitro and in vivo. In addition, injection of Stat3 decoy into skin tumors inhibited their growth. To our knowledge, these data provide the first evidence that Stat3 is required for de novo epithelial carcinogenesis, through maintaining the survival of DNA-damaged stem cells and through mediating and maintaining the proliferation necessary for clonal expansion of initiated cells during tumor promotion. Collectively, these data suggest that, in addition to its emerging role as a target for cancer therapy, Stat3 may also be a target for cancer prevention strategies.

Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate a keratinocyte-specific null mutation of Pten in mice (k5Pten(flox/flox) mice). k5Pten(flox/flox) mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, shaggy, and curly hair. Histological examination revealed that skin morphogenesis is accelerated in k5Pten(flox/flox) mice. Within 3 weeks of birth, 90% of k5Pten(flox/flox) mice die of malnutrition possibly caused by hyperkeratosis of the esophagus. All k5Pten(flox/flox) mice develop spontaneous tumors within 8.5 months of birth, and chemical treatment accelerates the onset of tumors. k5Pten(flox/flox) keratinocytes are hyperproliferative and resistant to apoptosis and show increased activation of the Pten downstream signaling mediators Akt/protein kinase B (PKB) and extracellular signal-regulated kinase. Pten is thus an important regulator of normal development and oncogenesis in the skin.

Ahnak/Desmoyokin is dispensable for proliferation, differentiation, and maintenance of integrity in mouse epidermis.

Desmoyokin was first isolated from bovine muzzle epidermis and thought to be an epidermal desmosome-related protein. We previously demonstrated that the Desmoyokin gene is identical to the Ahnak gene, which is expressed ubiquitously and downregulated in neuroblastomas. It was assumed Ahnak/Desmoyokin was associated with epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and embryonic development. To determine the precise biological function of Ahnak/Desmoyokin, we generated a null mutation in ES cells and mice. The resultant Ahnak/Desmoyokin-deficient ES cells normally differentiated into embryoid bodies and neural cells. The mutant mice were viable and fertile and showed no gross developmental defects. Electron microscopic examination of skin sections demonstrated that the ultrastructure of epidermal intercellular junctions, including desmosomes, of the mutant mice was indistinguishable from that of wild-type mice. Two-stage chemical skin carcinogenesis experiments showed no difference in frequency or onset of cutaneous tumor formation between wild-type and mutant mice. Moreover, no tumorigenesis was observed in other tissues and organs of mutant mice up to 2 y of age. These results lead us to conclude that Ahnak/Desmoyokin deficiency has only a minimal effect on epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and overall mouse development.

Involvement of connective tissue-type mast cells in Th1 immune responses via Stat4 expression.

Mast cells are the sentinels of immune systems and, like other immuno-competent cells, they are produced by hematopoietic stem cells. We analyzed the expression of signal transducer and activator of transcription 4 (Stat4), and investigated its role in mast cells. Murine mast cells are usually divided into 2 distinct populations by their distribution and contents of their granules: mucosal mast cells (MMCs) and connective tissue-type mast cells (CTMCs). Stat4 protein was detected in CTMCs but not in MMCs. The absence of Stat4 expression in cultured mast cells was due to the presence of Stat6. In T-helper (Th) cells, Stat4 plays an important role in Th1 shift by inducing a set of genes, such as interferon gamma (IFN-gamma) and interleukin-18 receptor alpha subunit (IL-18Ralpha). As in Th1 shift, we found that Stat4 trans-activated these genes in the Stat4-expressing cultured mast cells, namely, microphthalmia transcription factor (MITF)-deficient cultured MMCs, Stat6-deficient cultured MMCs, and cultured CTMCs. Stat4 also enhanced expression of nitric oxide synthase 2 (NOS2) in CTMCs, which brought about increased levels of NO-dependent cytotoxic activity. These data indicate that expression of Stat4 in CTMCs plays an important role on Th1 immune responses.

Reduced expression of IL-12 receptor beta2 and IL-18 receptor alpha genes in natural killer cells and macrophages derived from B6-mi/mi mice.

The mi transcriptional factor (MITF) is a basic helix-loop-helix leucine zipper-type transcriptional factor. The mi mutant allele encodes an abnormal MITF, in which one out of four consecutive arginines is deleted in the basic domain. The VGA-9-tg (tg) allele is another mutant allele and considered to be a null mutant allele. C57BL/6 (B6)-mi/mi mice showed abnormal phenotypes of natural killer (NK) cells and macrophages, whereas B6-tg/tg mice did not. The expression levels of the genes for the interleukin-12 receptor (IL-12R) beta2 and IL-18Ralpha were reduced in both the NK cells and macrophages of B6-mi/mi mice, while the expression levels of the corresponding genes in B6-tg/tg mice were unaffected. The B6-mi/mi NK cells and B6-mi/mi macrophages showed impaired responses to stimulation with IL-12, IL-18, and IL-12 plus IL-18 stimulation. The abnormal NK cell and macrophage of B6-mi/mi mice appear to be due to decreased expression of the IL-12Rbeta2 and IL-18Ralpha genes.

Tumorigenesis facilitated by Pten deficiency in the skin: evidence of p53-Pten complex formation on the initiation phase.

Pten, a tumor suppressor gene, is mutated in various human cancers and in hereditary cancer syndromes, such as Cowden disease. We have previously developed a knockout mouse in which Pten is specifically disrupted in the skin, resulting in hyperproliferation and spontaneous tumorigenesis of the skin keratinocytes. In this study, we further clarified the effects of Pten deficiency in tumorigenesis, by using a two-step model in intact skin of Pten knockout mouse. Although the conventional protocol requires serial exposures to DMBA and TPA, mice deficient for Pten developed skin papilloma within 6 weeks after a single exposure to DMBA, indicating that loss of Pten has a tumor-promoting effect. Serial exposure to DMBA-TPA ointments produced 10-fold more papillomas in the skin of knockout mice than in the wild-type counterpart, suggesting an increased rate of initiation. Therefore, we precisely examined the effect of DMBA. This treatment was highly apoptotic in wild-type mice, whereas the number of apoptotic cells was diminished in Pten-deficient skin. Moreover, primary keratinocytes isolated from Pten-deficient mice were also resistant to the apoptotic effect of DMBA. The status of p53, Pten proteins and downstream targets of p53, such as p21, 14-3-3, and Reprimo, were also examined, and we found that accumulation of p53 protein and up-regulation of p53 targets were delayed in Pten-knockout skin. These observations suggest that Pten is involved in rapid recruitment of p53 in the tumor initiation phase.