Molecular mechanisms of mitochondrial DNA release and activation of the cGAS-STING pathway.

In addition to constituting the genetic material of an organism, DNA is a tracer for the recognition of foreign pathogens and a trigger of the innate immune system. cGAS functions as a sensor of double-stranded DNA fragments and initiates an immune response via the adaptor protein STING. The cGAS-STING pathway not only defends cells against various DNA-containing pathogens but also modulates many pathological processes caused by the immune response to the ectopic localization of self-DNA, such as cytosolic mitochondrial DNA (mtDNA) and extranuclear chromatin. In addition, macrophages can cause inflammation by forming a class of protein complexes called inflammasomes, and the activation of the NLRP3 inflammasome requires the release of oxidized mtDNA. In innate immunity related to inflammasomes, mtDNA release is mediated by macropores that are formed on the outer membrane of mitochondria via VDAC oligomerization. These macropores are specifically formed in response to mitochondrial stress and tissue damage, and the inhibition of VDAC oligomerization mitigates this inflammatory response. The rapidly expanding area of research on the mechanisms by which mtDNA is released and triggers inflammation has revealed new treatment strategies not only for inflammation but also, surprisingly, for neurodegenerative diseases such as amyotrophic lateral sclerosis.

The Protective Effect of Zebularine, an Inhibitor of DNA Methyltransferase, on Renal Tubulointerstitial Inflammation and Fibrosis.

Renal fibrosis, the final pathway of chronic kidney disease, is caused by genetic and epigenetic mechanisms. Although DNA methylation has drawn attention as a developing mechanism of renal fibrosis, its contribution to renal fibrosis has not been clarified. To address this issue, the effect of zebularine, a DNA methyltransferase inhibitor, on renal inflammation and fibrosis in the murine unilateral ureteral obstruction (UUO) model was analyzed. Zebularine significantly attenuated renal tubulointerstitial fibrosis and inflammation. Zebularine decreased trichrome, α-smooth muscle actin, collagen IV, and transforming growth factor-ß1 staining by 56.2%. 21.3%, 30.3%, and 29.9%, respectively, at 3 days, and by 54.6%, 41.9%, 45.9%, and 61.7%, respectively, at 7 days after UUO. Zebularine downregulated mRNA expression levels of matrix metalloproteinase (MMP)-2, MMP-9, fibronectin, and Snail1 by 48.6%. 71.4%, 31.8%, and 42.4%, respectively, at 7 days after UUO. Zebularine also suppressed the activation of nuclear factor-κB (NF-κB) and the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6, by 69.8%, 74.9%, and 69.6%, respectively, in obstructed kidneys. Furthermore, inhibiting DNA methyltransferase buttressed the nuclear expression of nuclear factor (erythroid-derived 2)-like factor 2, which upregulated downstream effectors such as catalase (1.838-fold increase at 7 days, p < 0.01), superoxide dismutase 1 (1.494-fold increase at 7 days, p < 0.05), and NAD(P)H: quinone oxidoreduate-1 (1.376-fold increase at 7 days, p < 0.05) in obstructed kidneys. Collectively, these findings suggest that inhibiting DNA methylation restores the disrupted balance between pro-inflammatory and anti-inflammatory pathways to alleviate renal inflammation and fibrosis. Therefore, these results highlight the possibility of DNA methyltransferases as therapeutic targets for treating renal inflammation and fibrosis.

Induction of DR5-Dependent Apoptosis by PGA2 through ATF4-CHOP Pathway.

Prostaglandin (PG) A2, a cyclopentenone PG, induced apoptosis in both HCT116 and HCT116 p53 -/- cells. Although PGA2-induced apoptosis in HCT116 cells was dependent on the p53-DR5 pathway, the mechanism underlying PGA2-induced apoptosis in HCT116 p53 -/- cells remains unknown. In this study, we observed that PGA2 caused an increase of mRNA expression of DR5 and protein expression even in HCT116 p53 -/- cells, accompanied by caspase-dependent apoptosis. Knockdown of DR5 expression by RNA interference inhibited PGA2-induced apoptosis in HCT116 p53 -/- cells. Parallel to the induction of apoptosis, PGA2 treatment upregulated expression of genes upstream of DR5 such as ATF4 and CHOP. Knockdown of CHOP prevented DR5-dependent cell death as well as the expression of DR5 protein. Furthermore, knockdown of ATF4 by RNA interference decreased both mRNA and protein levels of CHOP and DR5, thereby suppressing PGA2-induced cell death. Consistently, the DR5 promoter activity increased by PGA2 was not stimulated when the CHOP binding site in the DR5 promoter was mutated. These results collectively suggest that PGA2 may induce DR5-dependent apoptosis via the ATF4-CHOP pathway in HCT116 p53 null cells.

The Levels of Circulating MicroRNAs at 6-Hour Cardiac Arrest Can Predict 6-Month Poor Neurological Outcome.

Early prognostication in cardiac arrest survivors is challenging for physicians. Unlike other prognostic modalities, biomarkers are easily accessible and provide an objective assessment method. We hypothesized that in cardiac arrest patients with targeted temperature management (TTM), early circulating microRNA (miRNA) levels are associated with the 6-month neurological outcome. In the discovery phase, we identified candidate miRNAs associated with cardiac arrest patients who underwent TTM by comparing circulating expression levels in patients and healthy controls. Next, using a larger cohort, we validated the prognostic values of the identified early miRNAs by measuring the serum levels of miRNAs, neuron-specific enolase (NSE), and S100 calcium-binding protein B (S100B) 6 h after cardiac arrest. The validation cohort consisted of 54 patients with TTM. The areas under the curve (AUCs) for poor outcome were 0.85 (95% CI (confidence interval), 0.72-0.93), 0.82 (95% CI, 0.70-0.91), 0.78 (95% CI, 0.64-0.88), and 0.77 (95% CI, 0.63-0.87) for miR-6511b-5p, -125b-1-3p, -122-5p, and -124-3p, respectively. When the cut-off was based on miRNA levels predicting poor outcome with 100% specificity, sensitivities were 67.7% (95% CI, 49.5-82.6), 50.0% (95% CI, 32.4-67.7), 35.3% (95% CI, 19.7-53.5), and 26.5% (95% CI, 12.9-44.4) for the above miRNAs, respectively. The models combining early miRNAs with protein biomarkers demonstrated superior prognostic performance to those of protein biomarkers.

Inhibition of STAT3 enhances sensitivity to tamoxifen in tamoxifen-resistant breast cancer cells.

BACKGROUND: The mechanisms of endocrine resistance are complex, and deregulation of several oncogenic signalling pathways has been proposed. We aimed to investigate the role of the EGFR and Src-mediated STAT3 signalling pathway in tamoxifen-resistant breast cancer cells. METHODS: The ER-positive luminal breast cancer cell lines, MCF-7 and T47D, were used. We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. Cell viability was determined using an MTT assay, and protein expression levels were determined using western blot. Cell cycle and annexin V staining were analysed using flow cytometry. RESULTS: TamR cells showed decreased expression of estrogen receptor and increased expression of EGFR. TamR cells showed an acceleration of the G1 to S phase transition. The protein expression levels of phosphorylated Src, EGFR (Y845), and STAT3 was increased in TamR cells, while phosphorylated Akt was decreased. The expression of p-STAT3 was enhanced according to exposure time of tamoxifen in T47D cells, suggesting that activation of STAT3 can cause tamoxifen resistance in ER-positive breast cancer cells. Both dasatinib (Src inhibitor) and stattic (STAT3 inhibitor) inhibited cell proliferation and induced apoptosis in TamR cells. However, stattic showed a much stronger effect than dasatinib. Knockdown of STAT3 expression by siRNA had no effect on sensitivity to tamoxifen in MCF-7 cells, while that enhanced sensitivity to tamoxifen in TamR cells. There was not a significant synergistic effect of dasatinib and stattic on cell survival. TamR cells have low nuclear p21(Cip1) expression compared to MCF-7 cells and inhibition of STAT3 increased the expression of nuclear p21(Cip1) in TamR cells. CONCLUSIONS: The EGFR and Src-mediated STAT3 signalling pathway is activated in TamR cells, and inhibition of STAT3 may be a potential target in tamoxifen-resistant breast cancer. An increase in nuclear p21(Cip1) may be a key step in STAT3 inhibitor-induced cell death in TamR cells.

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis.

BACKGROUND: Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear. METHODS: Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys. RESULTS: Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice. CONCLUSION: These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Induction of p53-Dependent Apoptosis by Prostaglandin A2.

Prostaglandin (PG) A2, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA2 has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical pro-apoptotic genes, on PGA2-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA2 was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA2-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA2 induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-α (PFT-α), a chemical inhibitor of p53's transcriptional activity, interfered with the induction of apoptosis in PGA2-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA2-induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA2-induced apoptosis. In the meanwhile, in HCT116 p53-/- cells, PGA2 induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA2 in HCT116 p53-/- cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT- or NU7441. Collectively, these results suggest the following. PGA2 may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA2 may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/- cells.

Empagliflozin Contributes to Polyuria via Regulation of Sodium Transporters and Water Channels in Diabetic Rat Kidneys.

Besides lowering glucose, empagliflozin, a selective sodium-glucose cotransporter-2 (SGLT2) inhibitor, have been known to provide cardiovascular and renal protection due to effects on diuresis and natriuresis. However, the natriuretic effect of SGLT2 inhibitors has been reported to be transient, and long-term data related to diuretic change are sparse. This study was performed to assess the renal effects of a 12-week treatment with empagliflozin (3 mg/kg) in diabetic OLETF rats by comparing it with other antihyperglycemic agents including lixisenatide (10 µg/kg), a glucagon-like peptide receptor-1 agonist, and voglibose (0.6 mg/kg), an α-glucosidase inhibitor. At 12 weeks of treatment, empagliflozin-treated diabetic rats produced still high urine volume and glycosuria, and showed significantly higher electrolyte-free water clearance than lixisenatide or voglibose-treated diabetic rats without significant change of serum sodium level and fractional excretion of sodium. In empagliflozin-treated rats, renal expression of Na+-Cl- cotransporter was unaltered, and expressions of Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter, and epithelial Na+ channel were decreased compared with control diabetic rats. Empagliflozin increased an expression of aquaporin (AQP)7 but did not affect AQP3 and AQP1 protein expressions in diabetic kidneys. Despite the increased expression in vasopressin V2 receptor, protein and mRNA levels of AQP2 in empagliflozin-treated diabetic kidneys were significantly decreased compared to control diabetic kidneys. In addition, empagliflozin resulted in the increased phosphorylation of AQP2 at S261 through the increased cyclin-dependent kinases 1 and 5 and protein phosphatase 2B. These results suggest that empagliflozin may contribute in part to polyuria via its regulation of sodium channels and AQP2 in diabetic kidneys.

Inhibition of p300/CBP-Associated Factor Attenuates Renal Tubulointerstitial Fibrosis through Modulation of NF-kB and Nrf2.

p300/CBP-associated factor (PCAF), a histone acetyltransferase, is involved in many cellular processes such as differentiation, proliferation, apoptosis, and reaction to cell damage by modulating the activities of several genes and proteins through the acetylation of either the histones or transcription factors. Here, we examined a pathogenic role of PCAF and its potential as a novel therapeutic target in the progression of renal tubulointerstitial fibrosis induced by non-diabetic unilateral ureteral obstruction (UUO) in male C57BL/6 mice. Administration of garcinol, a PCAF inhibitor, reversed a UUO-induced increase in the renal expression of total PCAF and histone 3 lysine 9 acetylation and reduced positive areas of trichrome and α-smooth muscle actin and collagen content. Treatment with garcinol also decreased mRNA levels of transforming growth factor-ß, matrix metalloproteinase (MMP)-2, MMP-9, and fibronectin. Furthermore, garcinol suppressed nuclear factor-κB (NF-κB) and pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, whereas it preserved the nuclear expression of nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and levels of Nrf2-dependent antioxidants including heme oxygense-1, catalase, superoxide dismutase 1, and NAD(P)H:quinone oxidoreductase 1. These results suggest that the inhibition of inordinately enhanced PCAF could mitigate renal fibrosis by redressing aberrant balance between inflammatory signaling and antioxidant response through the modulation of NF-κB and Nrf2.

Thyrocyte-specific deletion of insulin and IGF-1 receptors induces papillary thyroid carcinoma-like lesions through EGFR pathway activation.

Insulin and insulin-like growth factor (IGF)-1 signaling in the thyroid are thought to be permissive for the coordinated regulation by thyroid-stimulating hormone (TSH) of thyrocyte proliferation and hormone production. However, the integrated role of insulin receptor (IR) and IGF-1 receptor (IGF-1R) in thyroid development and function has not been explored. Here, we generated thyrocyte-specific IR and IGF-1R double knockout (DTIRKO) mice to precisely evaluate the coordinated functions of these receptors in the thyroid of neonates and adults. Neonatal DTIRKO mice displayed smaller thyroids, paralleling defective folliculogenesis associated with repression of the thyroid-specific transcription factor Foxe1. By contrast, at postnatal day 14, absence of IR and IGF-1R paradoxically induced thyrocyte proliferation, which was mediated by mTOR-dependent signaling pathways. Furthermore, we found elevated production of TSH during the development of follicular hyperplasia at 8 weeks of age. By 50 weeks, all DTIRKO mice developed papillary thyroid carcinoma (PTC)-like lesions that correlated with induction of the ErbB pathway. Taken together, these data define a critical role for IR and IGF-1R in neonatal thyroid folliculogenesis. They also reveal an important reciprocal relationship between IR/IGF-1R and TSH/ErbB signaling in the pathogenesis of thyroid follicular hyperplasia and, possibly, of papillary carcinoma.

Effects of Oncostatin M on Invasion of Primary Trophoblasts under Normoxia and Hypoxia Conditions.

PURPOSE: To investigate the effect of oncostatin M (OSM) on protein expression levels and enzymatic activities of matrix metalloprotainase (MMP)-2 and MMP-9 in primary trophoblasts and the invasiveness thereof under normoxia and hypoxia conditions. MATERIALS AND METHODS: Protein expression levels and enzymatic activities of MMP-2 and MMP-9 in primary trophoblasts under normoxia and hypoxia conditions were examined by Western blot and zymography, respectively. Effects of exogenous OSM on the in vitro invasion activity of trophoblasts according to oxygen concentration were also determined. Signal transducer and activator of transcription 3 (STAT3) siRNA was used to determine whether STAT3 activation in primary trophoblasts was involved in the effect of OSM. RESULTS: OSM enhanced protein expression levels and enzymatic activities of MMP-2 and MMP-9 in term trophoblasts under hypoxia condition, compared to normoxia control (p<0.05). OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly suppressed by STAT3 siRNA silencing under normoxia and hypoxia conditions (p<0.05). Hypoxia alone or OSM alone did not significantly increase the invasiveness of term trophoblasts. However, the invasion activity of term trophoblasts was significantly increased by OSM under hypoxia, compared to that without OSM treatment under normoxia. CONCLUSION: OSM might be involved in the invasiveness of extravillous trophoblasts under hypoxia conditions via increasing MMP-2 and MMP-9 enzymatic activities through STAT3 signaling. Increased MMP-9 activity by OSM seems to be more important in primary trophoblasts.

Correction: Treatment combining aliskiren with paricalcitol is effective against progressive renal tubulointerstitial fibrosis via dual blockade of intrarenal renin.

[This corrects the article DOI: 10.1371/journal.pone.0181757.].

D­Pinitol alleviates cyclosporine A­induced renal tubulointerstitial fibrosis via activating Sirt1 and Nrf2 antioxidant pathways.

Although the mechanism of cyclosporine A (CsA)­induced renal injury remains to be fully elucidated, accumulating evidence suggests that oxidative stress is critical in producing CsA­induced structural and functional renal impairment. The present study investigated the effect of D­pinitol, a cyclitol present in soybean, on chronic CsA nephropathy. Male ICR mice were treated with vehicle, CsA (30 mg/kg/day), D­pinitol (50 mg/kg/day) or a combination of CsA and D­pinitol for 28 days. To assess which pathway responding to oxidative stress is augmented by D­pinitol, the expression levels of several antioxidant enzymes and their possible regulators were measured. Treatment with D­pinitol significantly suppressed the increase of serum creatinine and decrease of urine osmolality, compared with the CsA control group. Histological examination of Masson's trichrome­ and α­smooth muscle actin­stained renal tissue demonstrated that the CsA­induced tubulointerstitial fibrosis and inflammation were attenuated by D­pinitol. Following the administration of D­pinitol, there were increased expression levels of heme oxygenase­1, NAD(P)H:quinone oxidoreductase 1, superoxide dismutase 1 and catalase in CsA­treated kidneys. In addition, D­pinitol increased the level of sirtuin 1 (Sirt1), and the total and nuclear expression levels of nuclear erythroid factor 2­related factor 2 (Nrf2), suggesting that activation of the Sirt1 and Nrf2 pathways may induce the cellular antioxidant system against CsA­induced nephropathy. Collectively, these data suggested that D­pinitol may protect the kidney from CsA­induced fibrosis, and that this renoprotective effect of D­pinitol was due to the inhibition of oxidative stress through the activation of Sirt1 and Nrf2, and the subsequent enhancement of antioxidant enzymes.

Treatment combining aliskiren with paricalcitol is effective against progressive renal tubulointerstitial fibrosis via dual blockade of intrarenal renin.

The aim of this study was to assess any potential additive effects of a treatment combining aliskiren with paricalcitol on reducing renal fibrosis. C57BL/6J mice were treated individually with aliskiren and/or paricalcitol until 7 days after initiation of unilateral ureteral obstruction (UUO).In obstructed kidneys of UUO mice, monotherapy with aliskiren or paricalcitol significantly attenuated interstitial fibrosis, collagen IV accumulation, and α-smooth muscle actin- and terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling-positive cells. The combination treatment showed additive efficacy in inhibition of these parameters. Renal NADPH oxidase (Nox)1 and Nox2 were significantly decreased by aliskiren or paricalcitol alone or in combination, while renal Nox4 expression was significantly reduced by paricalcitol mono- or combination treatment. Increased levels of p-Erk and p-p38 MAPK, and NF-κB in UUO kidneys were also significantly reduced by either aliskiren or paricalcitol treatment alone or in combination. Aliskiren or paricalcitol monotherapy significantly reduced the expression of (pro)renin receptor in UUO kidneys. In addition, aliskiren tended to augment renin expression in UUO kidneys, but paricalcitol reduced its expression level. The combination treatment effectively blocked both (pro)renin receptor and renin expression induced by aliskiren, and resulted in a further reduction of the renal expression of angiotensin II AT1 receptor. Aliskiren failed to increase the expression of vitamin D receptor in UUO kidneys, but the combination treatment restored its expression level. Taken together, a treatment combining aliskiren with paricalcitol better inhibits UUO-induced renal injury. The mechanism of this synergy may involve more profound inhibition of the intrarenal renin-angiotensin system.

T-type calcium channel blocker attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis by activating the Nrf2 antioxidant pathway.

Besides its effect on high blood pressure, T-type calcium channel blocker is renoprotective in experimental models of renal fibrosis. However, the exact mechanism of T-type calcium channel blocker on tubulointerstitial fibrosis is unclear. We investigated whether the renoprotective effect of T-type calcium channel blocker is associated with modulation of the signaling of oxidative stress-induced renal fibrosis. Treatment with a non-hypotensive dose of efonidipine, a T-type calcium channel blocker, or nifedipine, an L-type channel blocker, was initiated one day before unilateral ureteral obstruction (UUO) in C57BL6/J mice, and was continued until 3 and 7 days after UUO. In the obstructed kidneys, treatment with efonidipine significantly attenuated interstitial fibrosis, collagen deposition and inflammation increased by UUO creation compared with treatment with nifedipine. Additionally, efonidipine significantly increased the expression of the antioxidant enzymes heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, catalase and superoxide dismutase 1. Increased apoptotic cell death and decreased B-cell lymphoma 2 expression were also significantly ameliorated by efonidipine. The expression of the histone acetyltransferase p300/CBP-associated factor, a regulator of inflammatory molecules, was significantly inhibited by efonidipine. These beneficial effects of efonipidine were attributed to the increased nuclear expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) on UUO day 3 and the increased expressions of both total and nuclear Nrf2 with elevated Kelch-like ECH-associated protein 1 on UUO day 7. The data indicate that T-type calcium channel blocker exerts beneficial effects in renal interstitial fibrosis by activating Nrf2 and subsequent antioxidant enzymes.

Identification of a novel GLA mutation (Y88C) in a Korean family with Fabry nephropathy: a case report.

BACKGROUND: Fabry disease is a rare X-linked lysosomal storage disorder caused by α-galactosidase A deficiency. With the advancement of molecular diagnostic tools, more disease-causing mutations in α-galactosidase A (GLA) have been identified in Fabry disease. We found a novel mutation in a Korean family with predominant renal manifestations of the disease. CASE PRESENTATION: A 24-year-old man who wanted to donate a kidney to his 28-year-old brother with end-stage renal disease of unknown cause was evaluated. The 24-year-old man underwent percutaneous renal biopsy because of an accidentally found proteinuria. Electron microscopy of his renal biopsy showed numerous electron-dense multi-lamellar inclusions in the epithelial cytoplasm, typical for Fabry disease. Clinical and laboratory evaluation including the assessment of GLA enzyme activity and direct DNA sequencing in four members of the family were performed. Renal biopsy findings in the two affected male patients were described. Re-evaluation of a renal biopsy specimen of his 28-year-old brother obtained when he was diagnosed with renal failure revealed a very focal area of suspicious multilamellated structures in the Bowman's space. DNA sequencing on the young man, his brother, and his mother revealed a novel GLA gene mutation, c.263A > G (p.Tyr88Cys). The three all showed decreased α-galactosidase A activity. CONCLUSION: A novel GLA mutation, c.263A > G (p.Tyr88Cys), was found in a Korean family with predominant renal manifestations of Fabry disease.

STAT3 and ERK Signaling Pathways Are Implicated in the Invasion Activity by Oncostatin M through Induction of Matrix Metalloproteinases 2 and 9.

PURPOSE: Our previous studies have shown that oncostatin M (OSM) promotes trophoblast invasion activity through increased enzyme activity of matrix metalloproteinase (MMP)-2 and -9. We further investigated OSM-induced intracellular signaling mechanisms associated with these events in the immortalized human trophoblast cell line HTR8/SVneo. MATERIALS AND METHODS: We investigated the effects of OSM on RNA and protein expression of MMP-2 and -9 in the first-trimester extravillous trophoblast cell line (HTR8/SVneo) via Western blot. The selective signal transducer and activator of transcription (STAT)3 inhibitor, stattic, STAT3 siRNA, and extracellular signal-regulated kinase (ERK) siRNA were used to investigate STAT3 and ERK activation by OSM. The effects of STAT3 and ERK inhibitors on OSM-induced enzymatic activities of MMP-2 and -9 and invasion activity were further determined via Western blot and gelatin zymography. RESULTS: OSM-induced MMP-2 and -9 protein expression was significantly suppressed by STAT3 inhibition with stattic and STAT3 siRNA silencing, whereas the ERK1/2 inhibitor (U0126) and ERK silencing significantly suppressed OSM-induced MMP-2 protein expression. OSM-induced MMP-2 and MMP-9 enzymatic activities were significantly decreased by stattic pretreatment. The increased invasion activity induced by OSM was significantly suppressed by STAT3 and ERK1/2 inhibition, though to a greater extent by STAT3 inhibition. CONCLUSION: Both STAT3 and ERK signaling pathways are involved in OSM-induced invasion activity of HTR8/SVneo cells. Activation of STAT3 appears to be critical for the OSM-mediated increase in invasiveness of HTR8/SVneo cells.

Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers.

WISP3 is involved in many cancer-related processes including epithelial-mesenchymal transition, cell death, invasion, and metastasis and is considered a tumor suppressor. The aim of our study was to find whether WISP3 gene was mutated and expressionally altered in gastric (GC) and colorectal cancers (CRCs). WISP3 gene possesses a mononucleotide repeat in the coding sequence that could be mutated in cancers with high microsatellite instability (MSI-H). We analyzed 79 GCs and 156 CRCs, and found that GCs (8.8%) and CRCs (10.5%) with MSI-H, but not those with microsatellite stable/low MSI, harbored a frameshift mutation. We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutation in 16 CRCs and found that the WISP3 mutation exhibited regional ITH in 25% of the CRCs. In immunohistochemistry, loss of WISP3 expression was identified in 24% of GCs and 21% of CRCs. The loss of expression was more common in those with WISP3 mutation than with wild-type WISP3 and those with MSI-H than with microsatellite stable/low MSI. Our data indicate that WISP3 harbored not only frameshift mutation but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of GC and CRC with MSI-H by inhibiting tumor suppressor functions of WISP3. Our data also suggest that mutation analysis in multiregions is needed for a proper evaluation of mutation status in GC and CRC with MSI-H.

Deletion of IGF-1 Receptors in Cardiomyocytes Attenuates Cardiac Aging in Male Mice.

IGF-1 receptor (IGF-1R) signaling is implicated in cardiac hypertrophy and longevity. However, the role of IGF-1R in age-related cardiac remodeling is only partially understood. We therefore sought to determine whether the deletion of the IGF-1R in cardiomyocytes might delay the development of aging-associated myocardial pathologies by examining 2-year-old male cardiomyocyte-specific IGF-1R knockout (CIGF1RKO) mice. Aging was associated with the induction of IGF-1R expression in hearts. Cardiomyocytes hypertrophied with age in wild-type (WT) mice. In contrast, the cardiac hypertrophic response associated with aging was blunted in CIGF1RKO mice. Concomitantly, fibrosis was reduced in aged CIGF1RKO compared with aged WT hearts. Expression of proinflammatory cytokines such as IL-1α, IL-1ß, IL-6, and receptor activator of nuclear factor-κB ligand was increased in aged WT hearts, but this increase was attenuated in aged CIGF1RKO hearts. Phosphorylation of Akt was increased in aged WT, but not in aged CIGF1RKO, hearts. In cultured cardiomyocytes, IGF-1 induced senescence as demonstrated by increased senescence-associated ß-galactosidase staining, and a phosphoinositide 3-kinase inhibitor inhibited this effect. Furthermore, inhibition of phosphoinositide 3-kinase significantly prevented the increase in IL-1α, IL-1ß, receptor activator of nuclear factor-κB ligand, and p21 protein expression by IGF-1. These data reveal an essential role for the IGF-1-IGF-1R-Akt pathway in mediating cardiomyocyte senescence.