PARP1-mediated necrosis is dependent on parallel JNK and Ca²âº/calpain pathways.

Poly(ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme that can trigger caspase-independent necrosis. Two main mechanisms for this have been proposed: one involving RIP1 and JNK kinases and mitochondrial permeability transition (MPT), the other involving calpain-mediated activation of Bax and mitochondrial release of apoptosis-inducing factor (AIF). However, whether these two mechanisms represent distinct pathways for PARP1-induced necrosis, or whether they are simply different components of the same pathway has yet to be tested. Mouse embryonic fibroblasts (MEFs) were treated with either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ß-Lapachone, resulting in PARP1-dependent necrosis. This was associated with increases in calpain activity, JNK activation and AIF translocation. JNK inhibition significantly reduced MNNG- and ß-Lapachone-induced JNK activation, AIF translocation, and necrosis, but not calpain activation. In contrast, inhibition of calpain either by Ca(2+) chelation or knockdown attenuated necrosis, but did not affect JNK activation or AIF translocation. To our surprise, genetic and/or pharmacological inhibition of RIP1, AIF, Bax and the MPT pore failed to abrogate MNNG- and ß-Lapachone-induced necrosis. In conclusion, although JNK and calpain both contribute to PARP1-induced necrosis, they do so via parallel mechanisms.

Genetic manipulation of the cardiac mitochondrial phosphate carrier does not affect permeability transition.

The Mitochondrial Permeability Transition (MPT) pore is a voltage-sensitive unselective channel known to instigate necrotic cell death during cardiac disease. Recent models suggest that the isomerase cyclophilin D (CypD) regulates the MPT pore by binding to either the F0F1-ATP synthase lateral stalk or the mitochondrial phosphate carrier (PiC). Here we confirm that CypD, through its N-terminus, can directly bind PiC. We then generated cardiac-specific mouse strains overexpressing or with decreased levels of mitochondrial PiC to assess the functionality of such interaction. While PiC overexpression had no observable pathologic phenotype, PiC knockdown resulted in cardiac hypertrophy along with decreased ATP levels. Mitochondria isolated from the hearts of these mouse lines and their respective non-transgenic controls had no divergent phenotype in terms of oxygen consumption and Ca(2+)-induced MPT, as assessed by swelling and Ca(2+)-retention measurements. These results provide genetic evidence indicating that the mitochondrial PiC is not a critical component of the MPT pore.

Cardiac-specific hexokinase 2 overexpression attenuates hypertrophy by increasing pentose phosphate pathway flux.

BACKGROUND: The enzyme hexokinase-2 (HK2) phosphorylates glucose, which is the initiating step in virtually all glucose utilization pathways. Cardiac hypertrophy is associated with a switch towards increased glucose metabolism and decreased fatty acid metabolism. Recent evidence suggests that the increased glucose utilization is compensatory to the down-regulated fatty acid metabolism during hypertrophy and is, in fact, beneficial. Therefore, we hypothesized that increasing glucose utilization by HK2 overexpression would decrease cardiac hypertrophy. METHODS AND RESULTS: Mice with cardiac-specific HK2 overexpression displayed decreased hypertrophy in response to isoproterenol. Neonatal rat ventricular myocytes (NRVMs) infected with an HK2 adenovirus similarly displayed decreased hypertrophy in response to phenylephrine. Hypertrophy increased reactive oxygen species (ROS) levels, which were attenuated by HK2 overexpression, thereby decreasing NRVM hypertrophy and death. HK2 appears to modulate ROS via the pentose phosphate pathway, as inhibition of glucose-6-phosphate dehydrogenase with dehydroepiandrosterone decreased the ability of HK2 to diminish ROS and hypertrophy. CONCLUSIONS: These results suggest that HK2 attenuates cardiac hypertrophy by decreasing ROS accumulation via increased pentose phosphate pathway flux.

The mitochondrial protein C1qbp promotes cell proliferation, migration and resistance to cell death.

Complement 1q-Binding Protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration, and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation, and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP, and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels, and decreased cell migration compared to control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than anti-tumor, role for C1qbp, and indicate that C1qbp could serve as a molecular target for cancer therapeutics.