Angiopoietin-like protein 2 induces proinflammatory responses in peritoneal cells.

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1ß, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1ß, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.

Ectopic coexpression of keratin 8 and 18 promotes invasion of transformed keratinocytes and is induced in patients with cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) results from transformation of epidermal keratinocytes. Invasion of transformed keratinocytes through the basement membrane into the dermis results in invasive cSCC with substantial metastatic potential. To better understand the mechanisms for invasion and metastasis, we compared the protein expression profiles of a non-metastatic transformed mouse keratinocyte line and its metastatic derivative. Keratin 8 (Krt8) and Krt18, not seen in normal keratinocytes, were coexpressed and formed Krt8/18 filaments in the metastatic line. The metastatic line efficiently invaded an artificial basement membrane in vitro owing to the Krt8/18-coexpression, since coexpression of exogenous Krt8/18 in the non-invasive parental line conferred invasiveness. To test whether the Krt8/18-coexpression is induced and is involved in cSCC invasion, we examined specimens from 21 pre-invasive and 24 invasive cSCC patients by immunohistochemistry, and the ectopic Krt8/18-coexpression was almost exclusively found in invasive cSCC. Further studies are needed to examine the clinical significance of ectopic Krt8/18-coexpression in cSCC.

Rap2 function requires palmitoylation and recycling endosome localization.

Rap2A, Rap2B, and Rap2C are Ras-like small G proteins. The role of their post-translational processing has not been investigated due to the lack of information on their downstream signaling. We have recently identified the Traf2- and Nck-interacting kinase (TNIK), a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases, as a specific Rap2 effector. Here we report that, in HEK293T cells, Rap2A (farnesylated) and Rap2C (likely farnesylated), but not Rap2B (geranylgeranylated), require palmitoylation for membrane-association and TNIK activation, whereas all Rap2 proteins, including Rap2B, require palmitoylation for induction of TNIK-mediated phenotype, the suppression of cell spreading. Furthermore, we report for the first time that, in COS-1 cells, Rap2 proteins localize, and recruit TNIK, to the recycling endosomes, but not the Golgi nor the endoplasmic reticulum, in a palmitoylation-dependent manner. These observations implicate the involvement of palmitoylation and recycling endosome localization in cellular functions of Rap2 proteins.

MINK is a Rap2 effector for phosphorylation of the postsynaptic scaffold protein TANC1.

Rap1 and Rap2 are similar Ras-like G proteins but perform distinct functions. By the affinity chromatography/mass-spectrometry approach and the yeast two-hybrid screening, we identified Misshapen/NIKs-related kinase (MINK) as a novel Rap2-interacting protein that does not interact with Rap1 or Ras. MINK is a member of the STE20 group of mitogen-activated protein kinase kinase kinase kinases. The interaction between MINK and Rap2 was GTP-dependent and required Phe39 within the effector region of Rap2; the corresponding residue in Rap1 and Ras is Ser. MINK was enriched in the brain, and both MINK and its close relative, Traf2- and Nck-interacting kinase (TNIK), interacted with a postsynaptic scaffold protein containing tetratricopeptide repeats, ankyrin repeats and a coiled-coil region (TANC1) and induced its phosphorylation, under control of Rap2 in cultured cells. These are novel actions of MINK and TNIK, and consistent with a role of MINK as a Rap2 effector in the brain.

Direct activation of fission yeast adenylyl cyclase by heterotrimeric G protein gpa2.

Genetic studies on Schizosaccharomyces pombe adenylyl cyclase (cyr1) have shown that its activity is positively regulated by a heterotrimetric G protein a subunit gpa2 and that the resulting increase in intracellular cAMP concentration causes inhibition of sexual development including mating and meiosis. However, molecular mechanism underlying this gpa2-dependent regulation of cyr1 remains to be clarified. Here, we show that gpa2 exhibits a direct and GTP-dependent binding to the Ras-associating domain (RAD) of cyr1, which is identified by a computer algorithm-based search of the cyr1 amino acid sequence. Overexpression of this RAD results in acceleration of the sexual development of fission yeast cells presumably by competitive sequestration of gpa2. Furthermore, cyr1 is activated in vitro by the addition of purified gpa2, which is converted to the active state by treatment with AlF4-. These results indicate a crucial role of the RAD as a direct binding site of gpa2 in activation of cyr1. Thus, RADs, which have been defined as a conserved motif shared among the Ras-family small G protein-associating domains, are for the first time shown to exhibit a functional association with a member of the heterotrimeric G proteins.

Impact of human papillomavirus genotype on response to treatment and survival in patients receiving radiotherapy for squamous cell carcinoma of the cervix.

To determine the clinical implications and prognostic value of the human papillomavirus (HPV) genotype, we evaluated the various HPV types in patients receiving radiotherapy for squamous cell carcinoma of the cervix. The study population included 113 invasive squamous cell carcinoma patients treated with radiation or chemoradiation between 1993 and 2002. The median age of the patients was 61 years. Tumors were classified by the International Federation of Gynecology and Obstetrics staging as stage IB in 11 patients, stage II in 39, stage III in 57 and stage IVA in 6 patients. To investigate HPV infection and its genotypes in the tumor specimens, L1 consensus PCR was performed followed by the direct nucleotide sequencing of the PCR products. Ninety-five samples (84.1%) were positive for HPV DNA. The most prevalent type was HPV-16 (34.7%). Poorer response to radiotherapy was observed in the patients with the HPV-16 genotype, in which 7 of the 33 patients had persistent disease. Only 1 of the 10 patients with HPV-58, 1 of the 5 with HPV-31 and 5 of the 10 patients with HPV-33 had a recurrence. The 5-year survival rate was 90, 80, 69.4 and 39% in the HPV-58, HPV-31, HPV-16 and HPV-33 type groups, respectively. Patients with HPV-31 and HPV-58 types were found to have better survival, whereas patients with the HPV-33 type experienced a higher risk of death. HPV genotyping may serve as a potential biomarker of response to radiation and prognosis in cervical carcinoma patients undergoing radio- or chemoradiotherapy.

PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector.

Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific signaling role is unclear. By yeast two-hybrid screening, we have found that the Caenorhabditis elegans ortholog of Rap2 interacts with a protein containing a Rho-GTPase-activating protein (RhoGAP) domain, ZK669.1a, whose human ortholog PARG1 exhibits RhoGAP activity in vitro. ZK669.1a and PARG1 share a homology region with previously unknown function, designated the ZK669.1a and PARG1 homology (ZPH) region. Here we show that the ZPH region of PARG1 mediates interaction with Rap2. PARG1 interacted with Rap2 in a GTP-dependent manner but not with Ras or Rap1. We also show that PARG1 and its mutant lacking the ZPH region induce typical cytoskeletal changes for Rho inactivation in fibroblasts. Rap2 suppressed this in vivo action of PARG1 but not that of the mutant PARG1. These results suggest that PARG1 is a putative specific effector of Rap2 to regulate Rho.

A genotype distribution of human papillomaviruses detected by polymerase chain reaction and direct sequencing analysis in a large sample of common warts in Japan.

There have been no large-scale epidemiological studies of human papillomavirus (HPV) genotype distribution of common warts in Japan. A total of 213 patients with common warts (104 males and 109 females) in Japan were studied to detect HPV genotype distribution by polymerase chain reaction (PCR) and direct sequencing analysis. The results were as follows: 94 HPV-1a (44.1%), 35 HPV-4 (16.4%), 30 HPV-65 (14.1%), 13 HPV-27 (6.1%), 13 HPV-2a (6.1%), 9 HPV-57b (4.22%), 3 HPV-16 (1.41%), 2 HPV-6a (0.94%), 2 HPV-63 (0.94%), and 1 case for each of HPV-3, -5, -5b, -7, -10, -21, -29, -47, -56, -57, -62, and -92 (0.47%, respectively). Four cases (1.88%) were found in which two different HPV types were detected within the lesions: one case of HPV-1a with HPV-16, one case of HPV-1a with HPV-65, one case of HPV-6a with HPV-8, and one case of HPV-65 with HPV-16. There were seven cases of mucosal types (3.3%), that is, two HPV-6a, three HPV-16, one HPV-56, and one HPV-62, and three cases of epidermodysplasia verruciformis (EV)-related types (1.41%), that is, one HPV-5, one HPV-5b (both of which belonged to a high-risk group), and one HPV-47 (which belonged to a low-risk group). To date, this is the largest sequencing-based study of HPV for common warts in Japan. It is said that common warts are induced predominantly by HPV-2, -27, and -57 in European population. However, the present results showed that in Japan they were induced mostly by HPV-1, -4, and -65. This suggests that regional differences in HPV genotype distribution may exist between European and Japanese populations.

A large case-control study of cervical cancer risk associated with human papillomavirus infection in Japan, by nucleotide sequencing-based genotyping.

Using nucleotide sequencing-based genotyping, we conducted a case-control study to examine cervical cancer risk associated with human papillomavirus (HPV) infection in a Japanese population. A consensus primer pair was used to amplify DNA from the L1 region of HPV by polymerase chain reaction (PCR). By PCR, 311 of 356 patients with cervical cancer and 333 of 3249 control individuals were positive for HPV. By the direct sequencing of PCR products, nucleotide sequences of 30 genotypes were obtained. A high incidence of type 52 and a low incidence of type 16 were characteristic of the control group. Odds ratios were estimated for 18 genotypes. Types 71, 90, and 91, previously uncharacterized, were classified as low-risk genotypes, which is consistent with predictions made on the basis of phylogeny. The present study is the first large case-control study of its kind to use nucleotide sequencing-based genotyping.

Critical function of the Ras-associating domain as a primary Ras-binding site for regulation of Saccharomyces cerevisiae adenylyl cyclase.

Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.

Persistence of human papillomavirus infection as a predictor for recurrence in carcinoma of the cervix after radiotherapy.

OBJECTIVE: This study was undertaken to examine a possible correlation between clearance or persistence of human papillomavirus (HPV) infection and radiation response in carcinoma of the cervix. STUDY DESIGN: We reviewed 97 patients with HPV-positive cervical squamous cell carcinoma (International Federation of Gynecology and Obstetrics [FIGO] stage IB-IVA) treated with radiotherapy. Examination of HPV DNA was performed by the polymerase chain reaction-based assay. RESULTS: Cervical HPV DNA cleared in 42 patients (43.3%) and persisted in 55 patients (56.7%) at the end of irradiation. All of 42 HPV-cleared patients (100.0%) and 50 of 55 HPV-persistent patients (90.9%) had complete response. Of 92 patients with complete response, 20 (21.7%) had local recurrence develop. The recurrence rate was significantly higher in HPV-persisted patients (34.0%) than in HPV-cleared patients (7.1%) ( P = .0016). Univariate analysis demonstrated the significant differences for both 5-year local disease-free survival (LDFS) and overall survival (OAS) between HPV-cleared and HPV-persisted groups. Multivariate analysis showed that persistence of HPV DNA was the most powerful independent predictor for LDFS and OAS compared with other prognostic factors, such as FIGO stage or node swelling. CONCLUSION: In HPV-positive cervical carcinoma, persistence of HPV DNA in the cervix at the end of irradiation was highly predictive of LDFS and OAS.