HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in retinal pigment epithelial cells.

Mechanical stimulation as a mimic of drusen formation in the eye increases the expression of angiogenic factors in retinal pigment epithelial (RPE) cells, but the underlying molecular mechanisms remain unclear. We investigated and characterized the effects of mechanical stimulation on the expression of angiogenic factors in RPE cells both in vitro and in a mouse model. Mechanical stimulation increased the expression of vascular endothelial growth factor (VEGF, encoded by VEGFA) and other angiogenesis-related genes in cultured RPE1 cells. The presence of hypoxia-inducible factor 1α (HIF-1α, encoded by HIF1A) was also increased, and both knockdown of HIF-1α and treatment with the HIF-1α inhibitor CAY10585 attenuated the effect of mechanical stimulation on angiogenesis factor gene expression. Signaling by the tyrosine kinase SRC and p38 mitogen-activated protein kinase was involved in HIF-1α activation and consequent angiogenesis-related gene expression induced by mechanical stimulation. Our results suggest that SRC-p38 and HIF-1α signaling are involved in the upregulation of angiogenic factors in RPE cells by mechanical stimulation. Such in vivo suppression of upregulated expression of angiogenesis-related genes by pharmacological inhibitors of HIF-1α suggests a new potential approach to the treatment of age-related macular degeneration.

Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Benzalkonium chloride-induced myofibroblastic transdifferentiation of Tenon's capsule fibroblasts is inhibited by coculture with corneal epithelial cells or by interleukin-10.

Benzalkonium chloride (BAC) is used as a preservative in eyedrops but induces subconjunctival fibrosis that can result in failure of glaucoma surgery. Tenon's capsule fibroblasts in subconjunctival tissue interact with the corneal epithelium through tear fluid. With the use of a coculture system, we have now investigated the effect of human corneal epithelial (HCE) cells on myofibroblastic transdifferentiation of human Tenon fibroblasts (HTFs) induced by BAC (5 × 10-6%). Immunofluorescence and immunoblot analyses revealed that the BAC-induced expression of α smooth muscle actin (αSMA) in HTFs was suppressed by coculture of these cells with HCE cells (p < 0.01). The concentration of interleukin-10 (IL-10) in culture supernatants of BAC-treated HTFs was increased by coculture with HCE cells (17.26-fold, vs. coculure, p < 0.001). Immunofluorescence and immunoblot analyses also showed that exogenous IL-10 (300 pg/ml) suppressed the BAC-induced expression of αSMA by 43.65% (p < 0.05) as well as the nuclear translocation of myocardin-related transcription factor-A (MRTF-A) by 39.32% (p < 0.01) in HTFs cultured alone. Our findings suggest that corneal epithelial cells may protect against subconjunctival fibrosis by maintaining IL-10 levels and preventing the MRTF-A-dependent transdifferentiation of HTFs into myofibroblasts.

Inhibition of epithelial-mesenchymal transition in retinal pigment epithelial cells by a retinoic acid receptor-α agonist.

Epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-ß2 (TGF-ß2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-ß2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-ß2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-ß2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation.

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

Replicative senescent human cells possess altered circadian clocks with a prolonged period and delayed peak-time.

Over the last decade, a wide array of evidence has been accumulated that disruption of circadian clock is prone to cause age-related diseases and premature aging. On the other hand, aging has been identified as one of the risk factors linked to the alteration of circadian clock. These evidences suggest that the processes of aging and circadian clock feedback on each other at the animal level. However, at the cellular level, we recently revealed that the primary fibroblast cells derived from Bmal1-/- mouse embryo, in which circadian clock is completely disrupted, do not demonstrate the acceleration of cellular aging, i.e., cellular senescence. In addition, little is known about the impact of cellular senescence on circadian clock. In this study, we show for the first time that senescent cells possess a longer circadian period with delayed peak-time and that the variability in peak-time is wider in the senescent cells compared to their proliferative counterparts, indicating that senescent cells show alterations of circadian clock. We, furthermore, propose that investigation at cellular level is a powerful and useful approach to dissect molecular mechanisms of aging in the circadian clock.

Multidirectional analyses of hepatic chronotoxicity induced by cadmium in mice.

The expression levels or activities of biological defense factors can fluctuate daily following biological rhythms. We have focused on the relationship between injection timing and the degree of toxicity of cadmium (Cd) to promote the concept of "chronotoxicology," which introduces chronobiology to the field of toxicology. Our previous studies have clearly indicated that Cd may be subject to chronotoxicity. In this report, to confirm the character of the Cd-induced chronotoxicity, we performed multidirectional examinations. Male C57BL/6J mice that received a single intraperitoneal injection of CdCl2 at ZT6 showed drastic hepatic injury estimated by histopathological analyses, i.e., nuclear condensations, fatty degenerations, and hemorrhages, but showed no injury when injected at ZT18. This difference was supported by several biochemical analyses that were indicators of hepatic injury (levels of alanine aminotransferase, aspartate aminotransferase, and malondialdehyde). The chronotoxicity of Cd was also observed in multiple strains (ICR and Balb/c), in a different injection route (subcutaneous), and in multiple injections (5 injections). Based on these results, we propose that chronotoxicology may provide important information not only for toxicology but also for occupational health, i.e., the importance of injection timing for toxicity evaluation tests, the reproducibility of animal experiments, and the improvement in the quality of risk assessments for night shift workers who may be exposed to toxic substances at various times of the day.

Mechanisms of cadmium-induced chronotoxicity in mice.

Biological defense factors show diurnal variations in their expression levels or activities. These variations can induce the different sensitivity to external toxicants of a day. We reported earlier that mice showed clear diurnal variation of cadmium (Cd)-induced toxicity, i.e., chronotoxicity. In this report, we investigated additional new evidences for the cadmium (Cd)-induced chronotoxicity, and considered the mechanisms contributed to this chronotoxicity. Male C57BL/6J mice were injected with CdCl2 (6.4 mg/kg, one shot) intraperitoneally at 6 different time points of a day (zeitgeber time (ZT); ZT2, ZT6, ZT10, ZT14, ZT18 or ZT22) followed by monitoring the mortality until 14 days after the injection. We observed extreme difference in survival numbers: surprisingly, all mice died at ZT2 injection while all mice survived at ZT18 injection. Moreover, in non-lethal dose of Cd (4.5 mg/kg), the values of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) used as indexes of hepatotoxicity markedly increased at ZT6 injection while mostly unchanged at ZT18 injection. To consider the mechanisms of this extreme diurnal variation, we examined biochemical studies and concluded that the diurnal variation was not caused by the differences in hepatic Cd level, basal hepatic metallothionein (MT) level, and induction level or induction speed of hepatic MT. We suggested that one of the candidate determination factors was glutathione. We believe that the "chronotoxicology" for metal toxicity may be classic, yet new viewpoint in modern toxicology field.

Influence of injection timing on severity of cadmium-induced testicular toxicity in mice.

Cadmium (Cd) is one of the endocrine disrupter and is a well-known testicular toxicant. Recently, we reported that Cd-induced mortality was markedly different by injection timing. In this report, we investigated whether severity of testicular toxicity was affected by injection timing of Cd. C57BL/6J mice (male, 7 w) were received single intraperitoneal injection of CdCl(2) (4.5 mg/kg) at zeitgeber time 6 (ZT6) or ZT18; these injection timings showed highest (ZT6) or lowest (ZT18) mortality in our previous study (Miura, 2012). After one week of the injection, several parameters for testicular toxicity such as epididymal sperm motility and numbers of sperm head both in cauda epididymidis and testis were measured. At ZT6 injection group, all parameters examined were significantly reduced compared to the control group. However, very interestingly, no significant changes were observed at ZT18 injection group. We obtained similar results by another experiment in which mice were received single subcutaneous injection of CdCl(2) (4 or 6 mg/kg) followed by measuring the parameters ten days after the injection. This diurnal variation was not contradictory to the result of the lethal toxicity which we showed earlier. Therefore, our results indicate that the testicular toxicity of Cd is also influenced by the injection timing.