Role of RIN1 on telomerase activity driven by EGF-Ras mediated signaling in breast cancer.

Epidermal growth factor (EGF)-receptor regulates several downstream signaling pathways upon EGF stimulation that involves cell proliferation, migration and invasion. Internalized EGF-receptor is either recycled or degraded, which fate is regulated in part by Ras interference 1 (RIN1). In this study, we tested the hypothesis that RIN1, a Ras effector protein and Rab5 guanine nucleotide exchange factor, controls several signaling molecules leading to the modulation of the telomerase activity; thus, allowing proper cell proliferation. We report that expression of RIN1 completely blocked proliferation of MCF-12 A and MCF-7 cells, while partially inhibited proliferation of MDA-MB-231 cells upon EGF stimulation. Furthermore, expression of the C-terminal region of RIN1 selectively plays a critical role in the inhibition of the proliferation of MDA-MB-231 cells. However, this inhibitory effect was specifically affected by the independent expression of RIN1:Vsp9 and RIN1:RA domains. Additionally, endogenous level of expression of RIN1 was decreased in metastatic MDA-MB-231 cells as compared with non-tumorigenic MCF-12 A cells. We observed that expression of RIN1:R94A mutant blocked the proliferation of MDA-MB-231 cells, while expression of RIN1:Y561F and RIN1:R629A mutants completely reversed the inhibitory effect of RIN1:WT. Consistent with our observations, we found that expression of RIN1:WT in MDA-MB-231 cells diminished both protein kinase B (AKT) and extracellular-signal-regulated kinase 1/2 (ERK1/2) activities while p38 mitogen-activated protein kinases (p38 MAPK) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) were unaffected, but it produced downregulation of cellular-myelocytomatosis (c-Myc), erythroblast transformation specific (Ets2) and signal transducer and activator of transcription 3 (Stat3) activities. Inversely, expression of high-mobility group box 1 (HMBG1) was inhibited whereas expression of forkhead box transcription factor 1 (FOXO1) was increased in cells expressing RIN1. Interestingly, expression of RIN1 blocked telomerase activity and human telomerase reverse transcriptase (hTERT) expression, which correlated with the downregulations of c-Myc, Ets-2 and Stat3 activation. Taken together these findings indicate that RIN1 is a critical player in the modulation of the telomerase activity as well as hTERT expression in MDA-MB-231 cells upon EGF stimulation.

Dehydroleucodine inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest.

Aberrant levels of preadipocyte differentiation, triggered by adipocyte hyperplasia and hypertrophy, results in the obesogenic phenotype. Obesity is a risk factor for several metabolic disorders. In this paper, dehydroleucodine inhibited the accumulation of lipid droplets and decreased the elevations of triglycerides, and this inhibitory effect occurred during the early stage of adipogenesis. Thus, not only did dehydroleucodine downregulate the expression of C/EBPα and PPARγ, it also strongly blocked the expression of C/EBPß, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. The proliferation of preadipocytes was dramatically suppressed when dehydroleucodine was added to the medium as early as 24 hr. These results indicate that dehydroleucodine may specifically affect mitotic clonal expansion to inhibit preadipocyte differentiation. Dehydroleucodine arrested the cell cycle at the G0 /G1 phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Additionally, dehydroleucodine decreased phosphorylation of Erk1/2 and Akt. Furthermore, dehydroleucodine downregulated expression of histone demethylase JMJD2B as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPß and PPARγ, respectively. Collectively, our results indicate that dehydroleucodine inhibits preadipocyte differentiation by blocking mitotic clonal expansion via cell cycle arrest, which may be mediated by regulation of selective histone methylation/demethylation in transcription activation during the early step of adipogenesis.

A Novel Insight into Dehydroleucodine Mediated Attenuation of Pseudomonas aeruginosa Virulence Mechanism.

Increasing resistance of Pseudomonas aeruginosa (P. aeruginosa) to conventional treatments demands the search for novel therapeutic strategies. In this study, the antimicrobial activity of dehydroleucodine (DhL), a sesquiterpene lactone obtained from Artemisia (A.) douglasiana, was screened against several pathogenic virulence effectors of P. aeruginosa. In vitro, minimum inhibitory concentration of DhL was determined against P. aeruginosa strains PAO1, PA103, PA14, and multidrug resistant clinical strain, CDN118. Results showed that DhL was active against each strain where PAO1 and PA103 showed higher susceptibility (MIC 0.48 mg/mL) as compared to PA14 (MIC 0.96 mg/mL) and CDN118 (MIC 0.98 mg/mL). Also, when PAO1 strain was grown in the presence of DhL (MIC50, 0.12 mg/mL), a delay in the generation time was noticed along with significant inhibition of secretory protease and elastase activities, interruption in biofilm attachment phase in a stationary culture, and a significant decline in Type III effector ExoS. At MIC50, DhL treatment increased the sensitivity of P. aeruginosa towards potent antibiotics. Furthermore, treatment of P. aeruginosa with DhL prevented toxin-induced apoptosis in macrophages. These observations suggest that DhL activity was at the bacterial transcriptional level. Hence, antimicrobial activity of DhL may serve as leads in the development of new anti-Pseudomonas pharmaceuticals.

Rin1 is a negative regulator of the IL3 receptor signal transduction pathways.

Cytokines interact with cell-surface receptors, initiating signaling cascades that promote cell growth while inhibiting the pathways of apoptotic cells. Rin1 is a multifunctional protein that has been shown to regulate EGF receptor signaling and endocytosis. To examine the role of Rin1 in IL3 receptor signaling pathways, Rin1 and deletion mutants were expressed in cells using a retrovirus system. In this study, the overexpression of Rin1 molecules was shown to selectively block IL-3 activation of the Ras-Erk1/2 and PI3K/Akt pathways and the IL-3-stimulated incorporation of [3H] thymidine into DNA without a significant effect on the activity of the JNK and p38K pathways. Moreover, the depletion of Rin1 by RNA interference induced cell growth. In addition, Rin1 was also required as a downstream effector of BCR/ABL-induced cell proliferation. Interestingly, the expression of Rin1 selectively blocked the activation of Erk1/2 induced by the BCR/ABL oncogene. These results demonstrate that Rin1 plays an essential and selective role in both IL3- and BCR/ABL-induced cell proliferation and highlight a new function for Rin1 in leukemic cells.

Role of Rab5 in insulin receptor-mediated endocytosis and signaling.

Activated insulin receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between insulin receptor signaling and endocytosis is not well understood. This study utilizes both overexpression and depletion of Rab5 proteins to show that they play a critical role in both insulin-stimulated fluid phase and receptor-mediated endocytosis. Specifically, Rab5:WT and Rab5:Q79L (a GTP-hydrolysis defective mutant) enhance both types of endocytosis in response to insulin, while Rab5:S34N (a GTP-binding defective mutant) has the opposite effect. Morphological analysis indicates that both Rab5 and insulin receptor are found on early endosomes, but not at the plasma membrane. In addition, expression of Rab5:WT and Rab5:Q79L enhance both Erk1/2 and Akt activation without affecting JN- and p38-kinase activities, while the expression of Rab5:S34N blocks both Erk1/2 and Akt activation. Consistent with these observations, DNA synthesis is also altered by the expression of Rab5:S34N. Taken together, these results demonstrate that Rab5 is required for insulin receptor membrane trafficking and signaling.

Rin1 regulates insulin receptor signal transduction pathways.

Rin1 is a multifunctional protein containing several domains, including Ras binding and Rab5 GEF domains. The role of Rin1 in insulin receptor internalization and signaling was examined by expressing Rin1 and deletion mutants in cells utilizing a retrovirus system. Here, we show that insulin-receptor-mediated endocystosis and fluid phase insulin-stimulated endocytosis are enhanced in cells expressing the Rin1:wild type and the Rin1:C deletion mutant, which contain both the Rab5-GEF and GTP-bound Ras binding domains. However, the Rin1:N deletion mutant, which contains both the SH2 and proline-rich domains, blocked insulin-stimulated receptor-mediated and insulin-stimulated fluid phase endocytosis. In addition, the expression of Rin1:delta (429-490), a natural occurring splice variant, also blocked both receptor-mediated and fluid phase endocystosis. Furthermore, association of the Rin1 SH2 domain with the insulin receptor was dependent on tyrosine phosphorylation of the insulin receptor. Morphological analysis indicates that Rin1 co-localizes with insulin receptor both at the cell surface and in endosomes upon insulin stimulation. Interestingly, the expression of Rin1:wild type and both deletion mutants blocks the activation of Erk1/2 and Akt1 kinase activities without affecting either JN or p38 kinase activities. DNA synthesis and Elk-1 activation are also altered by the expression of Rin1:wild type and the Rin1:C deletion mutant. In contrast, the expression of Rin1:delta stimulates both Erk1/2 and Akt1 activation, DNA synthesis and Elk-1 activation. These results demonstrate that Rin1 plays an important role in both insulin receptor membrane trafficking and signaling.

Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis.

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.

The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization.

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.

Affinity sites for beta-glucuronidase on the surface of human spermatozoa.

Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that beta-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca(2+)-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and alpha-mannosidase from the Dictyostelium discoideum, suggesting that phosphomannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the beta-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.