This study examined the clinical significance of NAC1 and the expression level of its potential downstream target fatty acid synthase (FASN) in ovarian clear cell carcinomas (OCCCs), and evaluated the NAC1/FASN pathway as a potential therapeutic target.
NAC1 and FASN expression and NACC1 gene amplification were assessed in ovarian cancers by immunohistochemistry, fluorescence in situ hybridisation, and clinical data collected by a retrospective chart review. C75, a FASN inhibitor, was used to assess whether this pathway represented a therapeutic target in OCCC.
High NAC1 expression was most frequent in clear cell tumours (40.0%:24/60). NACC1 gene amplification was identified in none of the 58 OCCCs. The frequency of NACC1 gene amplification was significantly higher in the high-grade serous histology than in the clear cell histology (P<0.01). NAC1 expression was significantly correlated with FASN expression in both OCCC samples and OCCC cell lines. Either high NAC1 expression or high FASN expression significantly correlated with shorter progression-free and overall survival (P=0.002 and 0.0048). NAC1 overexpression stimulated FASN expression, and NAC1 silencing using siRNA decreased FASN expression in OCCC cell lines. Profound growth inhibition was observed in C75-treated carcinoma cells with FASN overexpression when compared with the response in carcinoma cells without FASN expression.
These findings indicate that NAC1/FASN overexpression is critical to the growth and survival of a subset of OCCC. The FASN silencing by the C75-induced phenotypes depends on the expression status of the targeted cell line. Therefore, NAC1/FASN pathway-targeted therapy may benefit selected OCCC patients.
ovarian clear cell carcinoma; fatty acid synthase; survival; NAC1; C75
Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC −α, −γ and −ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.
In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.
type II ovarian carcinoma; KRAS; MAPK1; gene amplification; survival; MEK inhibitor
Endometrial cancer is the fourth most common malignancy in women, with most cases being classified as early stage endometrioid tumors that carry a favorable prognosis. The endometrial serous histological subtype (ESC), however, while only accounting for 10% of all endometrial cancers is responsible for a disproportionate number of deaths. Unlike the estrogen-dependent, well differentiated endometrioid tumors, which are commonly associated with a younger age of onset, ESCs are estrogen-independent and tend to present at an advanced stage and in older women. Treatment for ESC entails aggressive surgery and multimodal adjuvant therapy. In this review, we describe the clinical behavior, molecular aspects, and treatment strategies for ESC.
endometrial serous carcinoma; endometrial carcinoma; Type II endometrial carcinoma; estrogen independent
We investigated the antiviral activity of nanosized copper(I) iodide (CuI) particles having an average size of 160 nm. CuI particles showed aqueous stability and generated hydroxyl radicals, which were probably derived from monovalent copper (Cu+). We confirmed that CuI particles showed antiviral activity against an influenza A virus of swine origin (pandemic [H1N1] 2009) by plaque titration assay. The virus titer decreased in a dose-dependent manner upon incubation with CuI particles, with the 50% effective concentration being approximately 17 μg/ml after exposure for 60 min. SDS-PAGE analysis confirmed the inactivation of the virus due to the degradation of viral proteins such as hemagglutinin and neuraminidase by CuI. Electron spin resonance (ESR) spectroscopy revealed that CuI generates hydroxyl radicals in aqueous solution, and radical production was found to be blocked by the radical scavenger N-acetylcysteine. Taken together, these findings indicate that CuI particles exert antiviral activity by generating hydroxyl radicals. Thus, CuI may be a useful material for protecting against viral attacks and may be suitable for applications such as filters, face masks, protective clothing, and kitchen cloths.
The aim of this study was to investigate the patterns of epidermal growth factor receptor (EGFR) overexpression, EGFR gene amplification, and the presence of activating mutations in the tyrosine kinase domain of this gene in squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas of the uterine cervix.
The EGFR expression, amplification, and mutation in cervical carcinomas were assessed by immunohistochemistry, fluorescence in situ hybridisation, and PCR–SSCP, respectively, and correlated with clinical data collected by a retrospective chart review. A functional assessment was performed by inactivating EGFR in cervical cancer cells with the potent inhibitor AG1478.
Immunohistochemical analysis revealed that 6 out of 59 (10.2%) cervical squamous cell carcinomas showed significant amplification of the EGFR locus, whereas none of the 52 adeno/adenosquamous cell carcinomas had detectable EGFR amplification (P<0.05). The EGFR amplification significantly correlated with shorter overall survival (P=0.001) in cervical squamous cell carcinomas. Multivariate analysis showed that EGFR gene amplification was an independent prognostic factor for overall survival (P=0.011). None of the squamous cell carcinomas (0%: 0 out of 32) had detectable oncogenic mutations in EGFR exons 18 through 21. The frequencies of KRAS and BRAF mutations were very low in both squamous and adeno/adenosquamous cell carcinomas. Sensitivity of cervical cancer cells to AG1478 depended on the presence of EGFR overexpression. AG1478-induced EGFR inactivation in cell lines with EGFR overexpression significantly suppressed tumour development and progression in a mouse xenograft model.
Our data suggest that EGFR signalling is important in a subset of cervical squamous cell carcinomas and that anti-EGFR therapy may benefit patients who carry the 7p11.2 amplicon in their tumours.
cervical cancer; EGFR; gene amplification; survival; squamous cell carcinoma; adenocarcinoma/adenosquamous carcinoma
The NFκB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFκB2/p100 precursor has been characterized as the fourth IκB type of suppressor for NFκB. However, the molecular mechanism(s) underlying regulated destruction of NFκB2 remains largely unknown. Here, we report that, unlike other IκBs, ubiquitination and destruction of NFκB2 are governed by SCFFbw7 in a GSK3-dependent manner. In Fbw7−/− cells, elevated expression of NFκB2/p100 leads to a subsequent reduction in NFκB signaling pathways and elevated sensitivity to TNFα-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFκBactivity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFκB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFκB2′s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFκB activity.
SCF-Skp2 E3 ubiquitin ligase (Skp2 hereafter) targets several cell cycle regulatory proteins for degradation via the ubiquitin-dependent pathway. However, the target-specific physiological functions of Skp2 have not been fully elucidated in kidney diseases. We previously reported an increase in Skp2 in progressive nephropathy and amelioration of unilateral ureteral obstruction (UUO) renal injury associated with renal accumulation of p27 in Skp2−/− mice. However, it remains unclear whether the amelioration of renal injury in Skp2−/− mice is solely caused by p27 accumulation, since Skp2 targets several other proteins. Using Skp2−/−p27−/− mice, we investigated whether Skp2 specifically targets p27 in the progressive nephropathy mediated by UUO. In contrast to the marked suppression of UUO renal injury in Skp2−/− mice, progression of tubular dilatation associated with tubular epithelial cell proliferation and tubulointerstitial fibrosis with increased expression of collagen and α-smooth muscle actin were observed in the obstructed kidneys in Skp2−/−p27−/− mice. No significant increases in other Skp2 target proteins including p57, p130, TOB1, cyclin A and cyclin D1 were noted in the UUO kidney in Skp2−/− mice, while p21, c-Myc, b-Myb and cyclin E were slightly increased. Contrary to the ameliorated UUO renal injure by Skp2-deficiency, the amelioration was canceled by the additional p27-deficiency in Skp2−/−p27−/− mice. These findings suggest a pathogenic role of the reduction in p27 targeted by Skp2 in the progression of nephropathy in UUO mice.
The cyclin-dependent kinase inhibitor (CKI) p57Kip2 plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27Kip1 at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.
Ovarian cancer is the most lethal gynecologic malignancy. Despite advances in chemotherapy, the five-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains around 30%. The most significant prognostic factor is stage, and most patients present at an advanced stage with peritoneal dissemination. There is often no clearly identifiable precursor lesion; therefore, the events leading to metastatic disease are poorly understood. This article reviews metastatic suppressor genes, the epithelial-mesenchymal transition (EMT), and the tumor microenvironment as they relate to ovarian cancer metastasis. Additionally, novel chemotherapeutic agents targeting the metastasis-related biochemical pathways are discussed.
cancer; metastasis suppressor gene; EMT; tumor microenvironment
Based on digital karyotyping, we have identified a new, discrete amplified region at ch19p13.2 in a high-grade ovarian serous carcinoma. To further characterize this region, we determined the frequency and biological significance of ch19p13.2 amplification by analyzing 341 high-grade serous carcinomas from The Cancer Genome Atlas (TCGA) and found an increased DNA copy number at this locus in 18% of cases. We correlated the DNA and RNA copy number by analyzing the TCGA dataset for all amplified genes and detected 7 genes within ch19p13.2 that were significantly correlated (R ≥0.54) and were, in fact, listed as the top 100 potential “driver” genes at a genome-wide scale. Interestingly, one of the 7 genes, NACC1, encoding NAC1 was previously reported to be involved in the development of tumor recurrence in ovarian serous carcinoma and to play a causal role in the development of paclitaxel resistance. Therefore, we selected NACC1 for validation in an independent cohort. Based on fluorescence in situ hybridization, we found that 35 (20%) of 175 high-grade serous carcinomas had an increased DNA copy number at the NACC1 locus, and those amplified cases were associated with early disease recurrence within 6 months (p= 0.013). A significantly high level of NAC1 protein expression based on immunohistochemistry was detected in amplified tumors as compared to non-amplified tumors (p< 0.005). In summary, our data suggest that amplification at the ch19p13.2 NACC1 locus, leading to NAC1 overexpression, is one of the molecular genetic alterations associated with early tumor recurrence in ovarian cancer.
E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box– and WD repeat domain–containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7–/– embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver.
Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied pRb targets partially inhibited tumorigenesis in Rb1+/- mice 1,2,3,4,5,6. Here, we report that inactivation of pRb target Skp2 7,8 completely prevents spontaneous tumorigenesis in Rb1+/- mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs, but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knockin mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCFSkp2 ubiquitin ligase as the underlying mechanism for Skp2’s essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.
The extracellular-regulated kinase (ERK) signaling pathway plays an important role in regulating the malignant potential of a cancer cell. However, the effect of ERK signaling on cancer metastasis is not clearly understood. In the present study, we examined the status of ERK activation in 88 ovarian carcinomas in order to clarify the clinicopathological and prognostic significance of phosphorylated ERK1/2 (p-ERK1/2). p-ERK1/2 expression was identified in 37 (42%) of 88 ovarian carcinomas. There was no significant correlation between p-ERK1/2 expression and any of the clinicopathological factors tested. No significant correlation between p-ERK1/2 expression and overall survival was found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.426). Next, to clarify the role of ERK1/2 activation in ovarian cancers, we inactivated ERK1/2 in ovarian cancer cells using the MEK inhibitor, CI-1040, which prevents ERK1/2 activation. Based on simulated wound healing and invasion chamber assays, we found that the motility and invasion of ES2 and MPSC1 cells with p-ERK1/2 were significantly reduced (P<0.01) after treatment with CI-1040. By contrast, CI-1040 did not have any effect on KF28 cells, which were negative for p-ERK1/2. Twist was down-regulated simultaneously with p-ERK1/2 following treatment of ES2 and MPSC1 cells with CI-1040. Immunohistochemistry of ovarian carcinoma tissue revealed that the increased expression of p-ERK1/2 significantly correlated with Twist expression (P<0.01). The findings in this study provide new insight into the biological role of ERK signaling in ovarian carcinomas. Additionally, our observations have an important therapeutic implication for patients with ovarian cancers that express p-ERK1/2 as these patients may potentially benefit from CI-1040 therapy.
ovarian carcinoma; phosphorylated-extracellular-regulated kinase 1/2; CI-1040; invasion
The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.
This study examined the status of KRAS and BRAF mutations, in relation to extracellular signal-regulated protein kinase (ERK) activation in 58 ovarian carcinomas to clarify the clinicopathological and prognostic significance of KRAS/BRAF mutations. Somatic mutations of either KRAS or BRAF were identified in 12 (20.6%) out of 58 ovarian carcinomas. The frequency of KRAS/BRAF mutations in conventional serous high-grade carcinomas (4.0% : 1/25) was significantly lower than that in the other histological type (32.3% : 10/31). Phosphorylated ERK1/2 (p-ERK1/2) expression was identified in 18 (38.2%) out of 45 ovarian carcinomas. KRAS/BRAF mutation was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage I, II (P<0.001), and p-ERK1/2 (P<0.001). No significant correlations between KRAS/BRAF mutations or p-ERK1/2 expression and overall survival were found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.2460, P=0.9339, respectively). Next, to clarify the roles of ERK1/2 activation in ovarian cancers harbouring KRAS or BRAF mutations, we inactivated ERK1/2 in ovarian cancer cells using CI-1040. Cl-1040 is a compound that selectively inhibits MAP kinase kinase (MEK), an upstream regulator of ERK1/2, and thus prevents ERK1/2 activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated cancer cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. This was evident in both in vitro and in vivo studies. The findings in this study indicate that an activated ERK1/2 pathway is critical to tumour growth and survival of ovarian cancers with KRAS or BRAF mutations. Furthermore, they suggest that the CI-1040-induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian cancers. Therefore, ovarian cancer patients with KRAS or BRAF mutations may benefit from CI-1040 treatment.
ovarian carcinoma; KRAS; BRAF; mutation; ERK1/2; CI-1040
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
Microwave endometrial ablation is a new, minimally invasive treatment option for menorrhagia. Its popularity in many countries is increasing due to its safety and simplicity.
We treated menorrhagia due to submucosal myomas in two patients with a modified microwave endometrial ablation device. Surgery was contraindicated in the first patient secondary to medical co-morbidities and in the second patient because of acute hemorrhagic shock. In both cases, the operation was highly effective and each patient was satisfied with her treatment outcome.
Given its safety, simplicity, and effectiveness, microwave endometrial ablation may be widely adopted for the emergent control of uterine bleeding in patients with poor surgical candidates.
MEA; Menorrhagia; Multiple systemic diseases; Acute hemorrhagic shock
The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).
Porphyromonas gingivalis; whole genome sequence; genome rearrangement; conjugative transposon; MITE
Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4+CD8+ stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4+CD8+ stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.
Wnt signaling regulates a variety of developmental processes in animals. Although the β-catenin dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here we delineate a pathway in which Wnt3a signals through the Gαq/11 subunits of G proteins to activate phosphatidylinositol signaling and PKCδ in the murine ST2 cells. The Gαq/11-PKCδ signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCδ homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCδ-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together these results reveal a novel Wnt-dependent osteogenic mechanism, and provide a potential target pathway for designing therapeutics to promote bone formation.
The nuclear export and cytoplasmic degradation of the cyclin-dependent kinase inhibitor p27 are required for effective progression of the cell cycle through the G0-G1 transition. The mechanism responsible for this translocation of p27 has remained unclear, however. We now show that cyclin D2 directly links growth signaling with the nuclear export of p27 at the G0-G1 transition in some cell types. The up-regulation of cyclin D2 in response to mitogenic stimulation was found to occur earlier than that of other D-type cyclins and in parallel with down-regulation of p27 at the G0-G1 transition. RNA interference-mediated depletion of cyclin D2 inhibited the nuclear export of p27 and delayed its degradation at the G0-G1 transition. In contrast, overexpression of cyclin D2 in G0 phase shifted the localization of p27 from the nucleus to the cytoplasm and reduced the stability of p27. Overexpression of the cyclin D2(T280A) mutant, whose export from the nucleus is impaired, prevented the translocation and degradation of p27. These results indicate that cyclin D2 translocates p27 from the nucleus into the cytoplasm for its KPC-dependent degradation at the G0-G1 transition.
Diabetes results from an inadequate mass of functional β cells, due to either β cell loss caused by immune assault or the lack of compensation to overcome insulin resistance. Elucidating the mechanisms that regulate β cell mass has important ramifications for fostering β cell regeneration and the treatment of diabetes. We report here that Skp2, a substrate recognition component of Skp1–Cul1–F-box (SCF) ubiquitin ligase, played an essential and specific role in regulating the cellular abundance of p27 and was a critical determinant of β cell proliferation. In Skp2–/– mice, accumulation of p27 resulted in enlarged polyploid β cells as a result of endoreduplication replacing proliferation. Despite β cell hypertrophy, Skp2–/– mice exhibited diminished β cell mass, hypoinsulinemia, and glucose intolerance. Increased insulin resistance resulting from diet-induced obesity caused Skp2–/– mice to become overtly diabetic, because β cell growth in the absence of cell division was insufficient to compensate for increased metabolic demand. These results indicate that the Skp2-mediated degradation pathway regulating the cellular degradation of p27 is essential for establishing β cell mass and to respond to increased metabolic demand associated with insulin resistance.
The gonads are responsible for the production of germ cells through both mitosis and meiosis. Skp2 is the receptor subunit of an SCF-type ubiquitin ligase and is a major regulator of the progression of cells into S phase of the cell cycle, which it promotes by mediating the ubiquitin-dependent degradation of p27, an inhibitor of cell proliferation. However, the role of the Skp2-p27 pathway in germ cell development remains elusive.
We now show that disruption of Skp2 in mice results in a marked impairment in the fertility of males, with the phenotypes resembling Sertoli cell-only syndrome in men. Testes of Skp2-/- mice manifested pronounced germ cell hypoplasia accompanied by massive apoptosis in spermatogenic cells. Flow cytometry revealed an increased prevalence of polyploidy in spermatozoa, suggesting that the aneuploidy of these cells is responsible for the induction of apoptosis. Disruption of the p27 gene of Skp2-/- mice restored germ cell development, indicating that the testicular hypoplasia of Skp2-/- animals is attributable to the antiproliferative effect of p27 accumulation.
Our results thus suggest that compromised cell cycle progression caused by the accumulation of p27 results in aneuploidy and the induction of apoptosis in gonadal cells of Skp2-/- mice. The consequent reduction in the number of mature gametes accounts for the decreased fertility of these animals. These findings reinforce the importance of the Skp2-p27 pathway in cell cycle regulation and in germ cell development.
Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a−/− mouse embryonic fibroblasts. Furthermore, Ufd2a+/− mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.