Prominin 1 is crucial for the early development of photoreceptor outer segments.

Prominin 1 (PROM1) is a pentaspan transmembrane glycoprotein localized on the nascent photoreceptor discs. Mutations in PROM1 are linked to various retinal diseases. In this study, we assessed the role of PROM1 in photoreceptor biology and physiology using the PROM1 knockout murine model (rd19). Our study found that PROM1 is essential for vision and photoreceptor development. We found an early reduction in photoreceptor response beginning at post-natal day 12 (P12) before eye opening in the absence of PROM1 with no apparent loss in photoreceptor cells. However, at this stage, we observed an increased glial cell activation, indicative of cell damage. Contrary to our expectations, dark rearing did not mitigate photoreceptor degeneration or vision loss in PROM1 knockout mice. In addition to physiological defects seen in PROM1 knockout mice, ultrastructural analysis revealed malformed outer segments characterized by whorl-like continuous membranes instead of stacked disks. In parallel to the reduced rod response at P12, proteomics revealed a significant reduction in the levels of protocadherin, a known interactor of PROM1, and rod photoreceptor outer segment proteins, including rhodopsin. Overall, our results underscore the indispensable role of PROM1 in photoreceptor development and maintenance of healthy vision.

Aberrant lipid accumulation and retinal pigmental epithelium dysfunction in PRCD-deficient mice.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd -/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and accumulation of neutral lipids in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd -/- mice exhibited age-related pathological features such as neutral lipid deposits, lipofuscin accumulation, Bruch's membrane (BrM) thickening and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd -/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd -/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.

Excessive tubulin glutamylation leads to progressive cone-rod dystrophy and loss of outer segment integrity.

Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors. The observed functional reduction was not due to cell death, levels, or the mislocalization of major phototransduction proteins. Instead, the increased tubulin glutamylation caused shortened photoreceptor axonemes and the formation of numerous abnormal membranous whorls that disrupted the integrity of photoreceptor outer segments (OS). Ultimately, excessive tubulin glutamylation led to the progressive loss of photoreceptors, affecting cones more severely than rods. Our results highlight the importance of maintaining tubulin glutamylation for normal photoreceptor function. Furthermore, we demonstrate that murine cone photoreceptors are more sensitive to disrupted tubulin glutamylation levels than rods, suggesting an essential role for axoneme in the structural integrity of the cone outer segment. This study provides valuable insights into the mechanisms of photoreceptor diseases linked to excessive tubulin glutamylation.

Prenylation is essential for the enrichment of cone phosphodiesterase-6 (PDE6) in outer segments and efficient cone phototransduction.

Phosphodiesterase-6 (PDE6) is the key phototransduction effector enzyme residing in the outer segment (OS) of photoreceptors. Cone PDE6 is a tetrameric protein consisting of two inhibitory subunits (γ') and two catalytic subunits (α'). The catalytic subunit of cone PDE6 contains a C-terminus prenylation motif. Deletion of PDE6α' C-terminal prenylation motif is linked to achromatopsia (ACHM), a type of color blindness in humans. However, mechanisms behind the disease and roles for lipidation of cone PDE6 in vision are unknown. In this study, we generated two knock-in mouse models expressing mutant variants of cone PDE6α' lacking the prenylation motif (PDE6α'∆C). We find that the C-terminal prenylation motif is the primary determinant for the association of cone PDE6 protein with membranes. Cones from PDE6α'∆C homozygous mice are less sensitive to light, and their response to light is delayed, whereas cone function in heterozygous PDE6α'∆C/+ mice is unaffected. Surprisingly, the expression level and assembly of cone PDE6 protein were unaltered in the absence of prenylation. Unprenylated assembled cone PDE6 in PDE6α'∆C homozygous animals is mislocalized and enriched in the cone inner segment and synaptic terminal. Interestingly, the disk density and the overall length of cone OS in PDE6α'∆C homozygous mutants are altered, highlighting a novel structural role for PDE6 in maintaining cone OS length and morphology. The survival of cones in the ACHM model generated in this study bodes well for gene therapy as a treatment option for restoring vision in patients with similar mutations in the PDE6C gene.

HSP90α is needed for the survival of rod photoreceptors and regulates the expression of rod PDE6 subunits.

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.

Loss of MPC1 reprograms retinal metabolism to impair visual function.

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.

Enoyl coenzyme a hydratase domain-containing 2, a potential novel regulator of myocardial ischemia injury.

BACKGROUND: We reported previously that Brown Norway (BN) rats are more resistant to myocardial ischemia/reperfusion (I/R) injury than are Dahl S (SS) rats. To identify the unique genes differentially expressed in the hearts of these rats, we used DNA microarray analysis and observed that enoyl coenzyme A hydratase-containing domain 2 (ECHDC2) is highly expressed (≈18-fold) in the SS hearts compared with the BN hearts. METHODS AND RESULTS: RT-PCR, Western blot, and immunohistochemistry analyses verified that ECHDC2 was highly expressed in SS hearts compared with the BN hearts. ECHDC2 gene locates at chromosome 5 of rat and is expressed in mitochondria of the heart, mainly in cardiomyocytes but not in cardiofibroblasts. Overexpression of ECHDC2 in cells increased susceptibility to I/R injury while knockdown of ECHDC2 enhanced resistance to I/R injury. Furthermore, we observed that left anterior descending coronary artery ligation-induced myocardial infarction was more severe in the SS hearts than in the BN hearts or SSBN5 hearts, which was built on SS rats but had the substitution of chromosome 5 from BN rats. We also demonstrated that ECHDC2 did not alter mitochondrial O2 consumption, metabolic intermediates and ATP production. By gas chromatography-mass spectrometry, we found that ECHDC2 overexpression increased the levels of the cellular branched chain amino acids leucine and valine. CONCLUSION: ECHDC2, a mitochondrial protein, may be involved in regulating cell death and myocardial injury. Its deficiency in BN rats contributes to their increased resistance to myocardial I/R compared with SS rats. ECHDC2 increases branched chain amino acid metabolism and appears to be a novel regulator linking cell metabolism with cardiovascular disease.

N-acetyl lysyltyrosylcysteine amide inhibits myeloperoxidase, a novel tripeptide inhibitor.

Myeloperoxidase (MPO) plays important roles in disease by increasing oxidative and nitrosative stress and oxidizing lipoproteins. Here we report N-acetyl lysyltyrosylcysteine amide (KYC) is an effective inhibitor of MPO activity. We show KYC inhibits MPO-mediated hypochlorous acid (HOCl) formation and nitration/oxidation of LDL. Disulfide is the major product of MPO-mediated KYC oxidation. KYC (≤4,000 µM) does not induce cytotoxicity in bovine aortic endothelial cells (BAECs). KYC inhibits HOCl generation by phorbol myristate acetate (PMA)-stimulated neutrophils and human promyelocytic leukemia (HL-60) cells but not superoxide generation by PMA-stimulated HL-60 cells. KYC inhibits MPO-mediated HOCl formation in BAEC culture and protects BAECs from MPO-induced injury. KYC inhibits MPO-mediated lipid peroxidation of LDL whereas tyrosine (Tyr) and tryptophan (Trp) enhance oxidation. KYC is unique as its isomers do not inhibit MPO activity, or are much less effective. Ultraviolet-visible spectral studies indicate KYC binds to the active site of MPO and reacts with compounds I and II. Docking studies show the Tyr of KYC rests just above the heme of MPO. Interestingly, KYC increases MPO-dependent H2O2 consumption. These data indicate KYC is a novel and specific inhibitor of MPO activity that is nontoxic to endothelial cell cultures. Accordingly, KYC may be useful for treating MPO-mediated vascular disease.

The protein partners of GTP cyclohydrolase I in rat organs.

OBJECTIVE: GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat. METHODS AND RESULTS: A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria. CONCLUSION: GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.

Cross talk between NADPH oxidase and autophagy in pulmonary artery endothelial cells with intrauterine persistent pulmonary hypertension.

Autophagy is a process for cells to degrade proteins or entire organelles to maintain a balance in the synthesis, degradation, and subsequent recycling of cellular products. Increased reactive oxygen species formation is known to induce autophagy. We previously reported that increased NADPH oxidase (NOX) activity in pulmonary artery endothelial cells (PAEC) from fetal lambs with persistent pulmonary hypertension (PPHN) contributes to impaired angiogenesis in PPHN-PAEC compared with normal PAEC. We hypothesized that increased NOX activity in PPHN-PAEC is associated with increased autophagy, which, in turn, contributes to impaired angiogenesis in PPHN-PAEC. In the present study, we detected increased autophagy in PPHN-PAEC as shown by increased ratio of the microtubule-associated protein 1 light chain (LC3)-II to LC3-I and increased percentage of green fluorescent protein-LC3 punctate positive cells. Inhibiting autophagy by 3-methyladenine, chloroquine, and beclin-1 knockdown in PPHN-PAEC has led to decreased autophagy and increased in vitro angiogenesis. Inhibition of autophagy also decreased the association between gp91(phox) and p47(phox), NOX activity, and superoxide generation. A nonspecific antioxidant N-acetylcysteine and a NOX inhibitor apocynin decreased autophagy in PPHN-PAEC. In conclusion, autophagy may contribute to impaired angiogenesis in PPHN-PAEC through increasing NOX activity. Our results suggest that, in PPHN-PAEC, a positive feedback relationship between autophagy and NOX activity may regulate angiogenesis.

Role of autophagy in angiogenesis in aortic endothelial cells.

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

Differential sensitivity to LPS-induced myocardial dysfunction in the isolated brown Norway and Dahl S rat hearts: roles of mitochondrial function, NF-κB activation, and TNF-α production.

Recently, we reported that Brown Norway (BN) rats were more resistant to lipopolysaccharide (LPS)-induced myocardial dysfunction than Dahl S (SS) rats. This differential sensitivity was exemplified by reduced production of proinflammatory cytokines and diminished nuclear factor-κB pathway activation. To further clarify the mechanisms of different susceptibility of these two strains to endotoxin, this study was designed to examine the alterations of cardiac and mitochondrial bioenergetics, proinflammatory cytokines, and signaling pathways after hearts were isolated and exposed to LPS ex vivo. Isolated BN and SS hearts were perfused with LPS (4 µg/mL) for 30 min in the Langendorff preparation. Lipopolysaccharide depressed cardiac function as evident by reduced left ventricular developed pressure and decreased peak rate of contraction and relaxation in SS hearts but not in BN hearts. These findings are consistent with our previous in-vivo data. Under complex I substrates, a higher oxygen consumption and hydrogen peroxide (H2O2) production were observed in mitochondria from SS hearts than those from BN hearts. Lipopolysaccharide significantly increased H2O2 levels in both SS and BN heart mitochondria; however, the increase in oxygen consumption and H2O2 production in BN heart mitochondria was much lower than that in SS heart mitochondria. In addition, LPS significantly decreased complex I activity in SS hearts but not in BN hearts. Furthermore, LPS induced higher levels of tumor necrosis factor-α and increased phosphorylation of IκκB and p65 more in SS hearts than in BN hearts. Our results clearly demonstrate that less mitochondrial dysfunction combined with a reduced production of tumor necrosis factor-α and diminished activation of nuclear factor-κB are involved in the mechanisms by which isolated BN hearts were more resistant to LPS-induced myocardial dysfunction.

Increased resistance to LPS-induced myocardial dysfunction in the Brown Norway rats versus Dahl S rats: roles of inflammatory cytokines and nuclear factor kappaB pathway.

We previously demonstrated that hearts from Brown Norway (BN) rats were more resistant to ischemic injury than hearts from Dahl S (SS) rats. Here we determined the susceptibility to LPS-induced cardiomyopathy in these rats and examined the involvement of inflammatory signaling. Both strains were treated with LPS (20 mg/kg) via i.p. injection for 6 h. Myocardial function was assessed by the Langendorff system, and proinflammatory cytokines were measured by the enzyme-linked immunosorbent assay. LPS significantly reduced left ventricular developed pressure in both strains. Interestingly, the decrease of left ventricular developed pressure in BN rat hearts was approximately 25% less than that in SS rat hearts. Furthermore, LPS significantly reduced the peak rate of contraction and the peak rate of relaxation in SS hearts but not in BN hearts. No differences in LPS-induced decreases in coronary flow rate were observed between BN and SS rats. In addition, LPS-induced increases in proinflammatory cytokines, TNF-alpha, IL-1beta, and IL-6, were significantly lower in both plasma and hearts of BN rats compared with production in SS rats. LPS notably up-regulated the expression of proinflammatory enzymes, iNOS and cyclooxygenase 2, in SS hearts but not in BN hearts. Interestingly, LPS did not stimulate Toll-like receptor 4 or its adaptor myeloid differentiation factor 88 expression in the hearts of either strain but did increase IkappaB and P65 phosphorylation, less prominently in BN hearts than in SS hearts. These data indicate that reduced production of proinflammatory cytokines and diminished nuclear factor kappaB activation are major mechanisms by which BN hearts are more resistant to LPS-induced myocardial dysfunction than SS hearts.

Identification and functional characterization of phosphorylation sites on GTP cyclohydrolase I.

OBJECTIVE: The posttranslational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites. METHODS AND RESULTS: Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167, and threonine (T) 231 in HEK293 cells, whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51, S72, T85, T91, T103, S130, S167 and T231). GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity. CONCLUSIONS: Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103, and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.

Inhibition of CDKS by roscovitine suppressed LPS-induced *NO production through inhibiting NFkappaB activation and BH4 biosynthesis in macrophages.

In inflammatory diseases, tissue damage is critically associated with nitric oxide ((*)NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (*)NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (*)NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (*)NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (*)NO production, iNOS, and COX-2 upregulation, activation of NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.

Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts.

Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10min and 24h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition of AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60min and lasted until 12h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.

AICAR inhibits proliferation and induced S-phase arrest, and promotes apoptosis in CaSki cells.

AIM: The aim of the present study was to determine the effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on proliferation, cell cycle, and apoptosis in the human epithelial cervical cancer cell line CaSki cells. METHODS: Cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to determine cell proliferation and viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and Annexin-V/propidium iodide staining were analyzed by fluorescence-activated cell sorting (FACS). A Western blot assay was used to evaluate the expression of AKT (also known as protein kinase B), mammalian target of rapamycin (mTOR), p53, and extracellular signal-regulated kinase (ERK). RESULTS: AICAR (500 micromol/L) significantly inhibited the proliferation of CaSki cells treated for 24, 48, and 72 h as determined by cell count. The cells at the G1 and G2 phases were dramatically decreased while cells at the S phase were increased in response to AICAR treatment for 24, 48, and 72 h. The MTT assay showed less viable cells and Hoechst fluorescent staining showed more apoptotic cells upon AICAR stimulation. The results of the Annexin-V staining demonstrated a time-dependent increase of apoptosis in cells treated with AICAR for 24, 36, and 48 h. Furthermore, AICAR activated caspase-3 in a time-dependent manner. It was also found that AICAR inhibited the phosphorylation of AKT and mTOR, which are important kinases regulating cell growth and survival. AICAR stimulation obviously increased the expression of the tumor suppressor p53 and the phosphorylation of ERK. CONCLUSION: AICAR inhibited proliferation and induced S phase arrest and promoted apoptosis in CaSki cells, which might be mediated by the downregulation of the AKT/mTOR pathway and the upregulation of the p53/ERK pathway.

Phenylarsine oxide inhibited beta-adrenergic receptor-mediated IL-6 secretion: inhibition of cAMP accumulation and CREB activation in cardiac fibroblasts.

As we previously reported, cAMP and p38 MAPK instead of protein kinase A were involved in beta-adrenergic receptor (beta-AR)-mediated interleukin-6 (IL-6) production in mouse cardiac fibroblasts. Besides kinases, phosphatases may also be involved in IL-6 gene regulation. To study the role of protein tyrosine phosphatases (PTPs) in beta-AR-mediated IL-6 production, we selected the most widely used PTP inhibitor, phenylarsine oxide (PAO). We found that PAO dose-dependently inhibited the IL-6 release in response to beta-AR agonist isoproterenol (ISO) in mouse cardiac fibroblasts. This effect was probably due to the inhibition of PTPs, resulting in increased tyrosine phosphorylation, since genistein, an inhibitor of protein tyrosine kinases further potentiated ISO-induced IL-6 production and could partially reverse the inhibitory effect of PAO. PAO also significantly inhibited the IL-6 production by forskolin, an adenylyl cyclase (AC) activator. Furthermore, PAO dose-dependently inhibited the increased cAMP accumulation by either ISO or forskolin and suppressed the phosphorylation of CREB, an important transcriptional factor for IL-6 gene expression. But PAO did not affect the activation of p38 MAPK by ISO. Although PAO was also reported to inhibit NADPH oxidase, the inhibition of NADPH oxidase by its specific inhibitor, diphenylene iodonium (DPI) could not suppress beta-AR-mediated IL-6 production, suggesting that NADPH oxidase may not contribute to the inhibitory effect of PAO on IL-6 production. To our knowledge, this is the first report that PAO can inhibit ISO-induced IL-6 expression and CREB phosphorylation, demonstrating that PTPs may negatively regulate beta-AR-mediated IL-6 production. This study may also further our understanding of beta-AR signaling and provide potential therapeutic targets for the treatment of heart diseases.