Rawad Hodeify

Vesicular traffic at the cell membrane regulates oocyte meiotic arrest

Vertebrate oocytes are maintained in meiotic arrest for prolonged periods of time before undergoing oocyte maturation in preparation for fertilization. Cyclic AMP (cAMP) signaling plays a crucial role in maintaining meiotic arrest, which is released by a species-specific hormonal signal. Evidence in both frog and mouse argues that meiotic arrest is maintained by a constitutively active G-protein coupled receptor (GPCR) leading to high cAMP levels. Because activated GPCRs are typically targeted for endocytosis as part of the signal desensitization pathway, we were interested in determining the role of trafficking at the cell membrane in maintaining meiotic arrest. Here we show that blocking exocytosis, using a dominant-negative SNAP25 mutant in Xenopus oocytes, releases meiotic arrest independently of progesterone. Oocyte maturation in response to the exocytic block induces the MAPK and Cdc25C signaling cascades, leading to MPF activation, germinal vesicle breakdown and arrest at metaphase of meiosis II with a normal bipolar spindle. It thus replicates all tested aspects of physiological maturation. Furthermore, inhibiting clathrin-mediated endocytosis hinders the effectiveness of progesterone in releasing meiotic arrest. These data show that vesicular traffic at the cell membrane is crucial in maintaining meiotic arrest in vertebrates, and support the argument for active recycling of a constitutively active GPCR at the cell membrane.

Gender differences control the susceptibility to ER stress-induced acute kidney injury.

Endoplasmic reticulum (ER) stress contributes to acute kidney injury induced by several causes. Kidney dysfunction was shown to be influenced by gender differences. In this study we observed differences in the severity of kidney injury between male and female mice in response to tunicamycin, an ER stress agent. Tunicamycin-treated male mice showed a severe decline in kidney function and extensive kidney damage of proximal tubules in the kidney outer cortex (S1 and S2 segments). Interestingly, female tunicamycin-treated mice did not show a decline in kidney function, and their kidneys showed damage localized primarily to proximal tubules in the inner cortex (S3 segment). Protein markers of ER stress, glucose-regulated protein, and X-box binding protein 1 were also more elevated in male mice. Similarly, the induction of apoptosis was higher in tunicamycin-treated male mice, as measured by the activation of Bax and caspase-3. Testosterone administered to female mice before tunicamycin resulted in a phenotype similar to male mice with a comparable decline in renal function, tissue morphology, and induction of ER stress markers. We conclude that kidneys of male mice are much more susceptible to ER stress-induced acute kidney injury than those of females. Moreover, this sexual dimorphism could provide an interesting model to study the relation between kidney function and injury to a specific nephron segment.

Ca2+ Homeostasis Regulates Xenopus Oocyte Maturation

Abstract In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocytes competence to undergo maturation in preparation for fertilization and embryonic development.

A possible mechanism of renal cell death after ischemia/reperfusion

In this issue of Kidney International, Linkermann, et al. provide the first evidence for a possible biochemical mechanism of necrotic kidney cell death associated with renal ischemia/reperfusion-induced acute kidney injury. The mechanisms of several pathways resulting in programmed necrosis were recently elucidated and rely on receptor-interacting protein kinases 1 and 3. Using an inhibitor of one of these kinases, Linkermann was able to ameliorate functional and morphologic kidney damage after ischemia/reperfusion.

Cdk2-dependent phosphorylation of p21 regulates the role of Cdk2 in cisplatin cytotoxicity

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21(WAF1/Cip1) was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21(S78D). Mutant p21(S78D) was an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

Increased expression of p21WAF1/CIP1 in kidney proximal tubules mediates fibrosis

Tissue fibrosis is a major cause of death in developed countries. It commonly occurs after either acute or chronic injury and affects diverse organs, including the heart, liver, lung, and kidney. Using the renal ablation model of chronic kidney disease, we previously found that the development of progressive renal fibrosis was dependent on p21(WAF1/Cip1) expression; the genetic knockout of the p21 gene greatly alleviated this disease. In the present study, we expanded on this observation and report that fibrosis induced by two different acute injuries to the kidney is also dependent on p21. In addition, when p21 expression was restricted only to the proximal tubule, fibrosis after injury was induced in the whole organ. One molecular fibrogenic switch we describe is transforming growth factor-β induction, which occurred in vivo and in cultured kidney cells exposed to adenovirus expressing p21. Our data suggests that fibrosis is p21 dependent and that preventing p21 induction after stress could be a novel therapeutic target.

Protection of cisplatin cytotoxicity by an inactive cyclin-dependent kinase

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

Effects of Hyperglycemia on Vascular Smooth Muscle Ca2

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different.