Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Identification and purification of a calcium-binding protein from Bacillus subtilis

A Ca(2+)-binding protein was identified in Bacillus subtilis in the log phase of growth. The molecular mass of this protein is about 38 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS and by gel filtration. The protein was found to be resistant 10 min at 65 degrees C and was purified about 400 times, starting from heated crude extract, by conventional procedures. This novel protein is able to bind Ca2+ in the presence of an excess of MgCl2 and KCl both in solution and after SDS gel electrophoresis and electrotransfer. Since an impairment of the Ca2+ intake, in Bacillus subtilis, results in an impairment of chemotactic behavior (Matsushita, T. et al (1988) FEBS lett. 236, 437-440), 38 kDa protein may be involved in the regulation of chemotaxis.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

Oxytocin stimulates in vitro angiogenesis via a Pyk-2/Src-dependent mechanism

We previously reported that the hypothalamic hormone oxytocin (OT), best known for its uterotonic activity, also stimulates migration and invasion in human umbilical vein endothelial cells (HUVECs), thus suggesting a possible role for the peptide in the regulation of angiogenesis. We identified the Gq coupling of OT receptors (OTRs) and phospholipase C (PLC) as the main effectors of OT's action in HUVECs. Moreover, the pro-migratory effect of OT required the OTR-induced activation of the phosphatidylinositol-3-kinase (PI-3-K)/AKT/endothelial nitric oxide synthase (eNOS) pathway. To better characterize the proposed pro-angiogenic effect of OT in HUVECs, we have now utilized a three-dimensional (3-D) in vitro angiogenesis assay, and demonstrated that OT stimulates the outgrowth of capillary-like structures from HUVEC spheroids to an extent comparable to that of vascular endothelial growth factor (VEGF). This OT effect was abolished by inhibitors of PLC, PI-3-K and Src kinase. It was also found that OT phosphorylates proline-rich tyrosine kinase-2 (Pyk-2) and Src kinase in a PLC- and calcium-dependent manner. Furthermore, knockdown of Pyk-2 expression by RNA interference markedly impaired Src phosphorylation, migration and endothelial cell sprouting induced by OT. In conclusion, by using a pharmacological and genetic approach, the OT pro-angiogenic action and the cascade of intracellular signals responsible for it were defined by showing for the first time that OT, by interacting with its Gq-coupled receptor, induces HUVEC capillary outgrowth via Pyk-2 phosphorylation, which activates Src which in turn activates the PI-3-K/AKT pathway.     

Aquaporin‐1 inhibition reduces metastatic formation in a mouse model of melanoma

Aquaporin‐1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis‐dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF‐1α, caspase‐3 (CASP3) and metalloproteinase‐2 (MMP2) protein levels. We found that AQP1 knock‐down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA‐treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1‐dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF‐1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.