Mitochondrial DNA replication stress triggers a pro-inflammatory endosomal pathway of nucleoid disposal.

Mitochondrial DNA (mtDNA) encodes essential subunits of the oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation. As a DAMP, mtDNA not only contributes to anti-viral resistance, but also causes pathogenic inflammation in many disease contexts. Cells experiencing mtDNA stress caused by depletion of the mtDNA-packaging protein, transcription factor A, mitochondrial (TFAM) or during herpes simplex virus-1 infection exhibit elongated mitochondria, enlargement of nucleoids (mtDNA-protein complexes) and activation of cGAS-STING innate immune signalling via mtDNA released into the cytoplasm. However, the relationship among aberrant mitochondria and nucleoid dynamics, mtDNA release and cGAS-STING activation remains unclear. Here we show that, under a variety of mtDNA replication stress conditions and during herpes simplex virus-1 infection, enlarged nucleoids that remain bound to TFAM exit mitochondria. Enlarged nucleoids arise from mtDNA experiencing replication stress, which causes nucleoid clustering via a block in mitochondrial fission at a stage when endoplasmic reticulum actin polymerization would normally commence, defining a fission checkpoint that ensures mtDNA has completed replication and is competent for segregation into daughter mitochondria. Chronic engagement of this checkpoint results in enlarged nucleoids trafficking into early and then late endosomes for disposal. Endosomal rupture during transit through this endosomal pathway ultimately causes mtDNA-mediated cGAS-STING activation. Thus, we propose that replication-incompetent nucleoids are selectively eliminated by an adaptive mitochondria-endosomal quality control pathway that is prone to innate immune system activation, which might represent a therapeutic target to prevent mtDNA-mediated inflammation during viral infection and other pathogenic states.

Rac1 mediates cadherin-11 induced cellular pathogenic processes in aortic valve calcification.

BACKGROUND: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated. APPROACH AND RESULTS: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions. CONCLUSION: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD.

Induction of aortic valve calcification by celecoxib and its COX-2 independent derivatives is glucocorticoid-dependent.

BACKGROUND: Celecoxib, a selective cyclooxygenase-2 inhibitor, was recently associated with increased incidence of aortic stenosis and found to produce a valvular calcification risk in vitro. Several cyclooxygenase-2 independent celecoxib derivatives have been developed and identified as possible therapies for inflammatory diseases due to their cadherin-11 inhibitory functions. Potential cardiovascular toxicities associated with these cyclooxygenase-2 independent celecoxib derivatives have not yet been investigated. Furthermore, the mechanism by which celecoxib produces valvular toxicity is not known. METHODS AND RESULTS: Celecoxib treatment produces a 2.8-fold increase in calcification in ex vivo porcine aortic valve leaflets and a more than 2-fold increase in calcification in porcine aortic valve interstitial cells cultured in osteogenic media. Its cyclooxygenase-2 independent derivative, 2,5-dimethylcelecoxib, produces a similar 2.5-fold increase in calcification in ex vivo leaflets and a 13-fold increase in porcine aortic valve interstitial cells cultured in osteogenic media. We elucidate that this offtarget effect depends on the presence of either of the two media components: dexamethasone, a synthetic glucocorticoid used for osteogenic induction, or cortisol, a natural glucocorticoid present at basal levels in the fetal bovine serum. In the absence of glucocorticoids, these inhibitors effectively reduce calcification. By adding glucocorticoids or hydrocortisone to a serum substitute lacking endogenous glucocorticoids, we show that dimethylcelecoxib conditionally induces a 3.5-fold increase in aortic valve calcification and osteogenic expression. Treatment with the Mitogen-activated protein kinase kinase inhibitor, U0126, rescues the offtarget effect, suggesting that celecoxib and dimethylcelecoxib conditionally augment Mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase activity in the presence of glucocorticoids. CONCLUSION: Here we identify glucocorticoids as a possible source of the increased valvular calcification risk associated with celecoxib and its cyclooxygenase-2 independent derivatives. In the absence of glucocorticoids, these inhibitors effectively reduce calcification. Furthermore, the offtarget effects are not due to the drug's intrinsic properties as dual cyclooxygenase-2 and cadherin-11 inhibitors. These findings inform future design and development of celecoxib derivatives for potential clinical therapy.

A Mouse Homolog of a Human TP53 Germline Mutation Reveals a Lipolytic Activity of p53.

The physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53178C/C mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and transactivated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development.