Since the discovery of L-arginine-nitric oxide (NO) pathway in control of cardiovascular function in 1980s, endothelial dysfunction characterized by decreased endothelial NO bioavailability is recognized as a critical hallmark of cardiovascular disease (CVD) and aging, L-arginine supplementation has been proposed as a potential therapeutic intervention primarily due to its role as the sole substrate for endothelial NO-synthase (eNOS). Although many experimental and clinical studies show improvement of endothelial functions by L-arginine supplementation, the beneficial effects could not be translated into the improvement of hard endpoint outcome. Even harmful effects such as worsening of endothelial function, cardiac function, and increased mortality in patients with advanced CVD are reported in the randomized clinical trials. Despite of the reported adverse effects in clinical trials, L-arginine intake is considered safe and is still widely used in our society to boost muscle mass in healthy and aged populations. While we believe that chronic L-arginine supplementation shall be carefully implemented in clinical settings or shall be avoided in patients with advanced CVD and aged population, future studies shall focus on investigation of the underlying mechanisms of the adverse effects of L-arginine supplementation. This is important not only for L-arginine supplementation but also for other amino acid supplementation aimed to improve cardiovascular health, particularly in the elderly population.

Abstract Background Circulating monocyte trans-endothelial migration is critical for vascular diseases. The monocyte surface proteins, i.e., integrins and membrane receptors are central in this process. Previous studies demonstrate that ablation of the mitochondrial arginase-2 ( ARG2 ) reduces monocyte/macrophage infiltration in cardiovascular disease. We further investigate whether ARG2 regulates integrin and surface receptor levels in monocytes and facilitates trans-endothelial migration, contributing to atherogenesis. Methods For this purpose, human THP1 cells deficient in ARG2 gene (THP1 ARG2–/– ) were generated by CRISPR-U ™ -mediated genome engineering. Atherosclerotic Apoe –/– Arg2 + / + mice and Apoe –/– Arg2 –/– mouse models are used. Proteomic profiling, cellular/molecular biology methods, and confocal microscopy were utilized for gene or protein expression analysis. Results As compared to the control THP1 WT cells, the THP1 ARG2–/– cells reveal decreased adhesion and trans-endothelial migratory activities towards chemoattractants in a transwell co-culture system, accompanied by lower levels of αL and α4 integrins, CD99 and PECAM1 (CD31) (surface molecules for trans-endothelial activity) and CCR2 (the chemoattractant receptor). The monocyte adhesion to endothelial cells is reduced by blocking LFA-1 (αLβ2) or VLA-4 (α4β1) integrin. Pro-inflammatory polarization of the THP1 cells with LPS does not affect the integrin and surface receptor levels, however, enhances release of several pro-inflammatory cytokines, which is reduced in THP1 ARG2–/– cells due to reduced TLR4–ERK–NF-κB signaling. Conditioned medium from the LPS-primed THP1 WT reveals higher capacity to enhance endothelial VCAM-1 and ICAM-1 levels than the THP1 ARG2–/– cells, that is partly mediated by IL-1β. Moreover, ARG2-dependent TGF-β signaling was found to selectively regulate αL expression in monocytes. Finally, as compared to Apoe –/– Arg2 + / + mice, the ApoE –/– Arg2 –/– mice show significantly decreased macrophage integrin, chemoattractant receptor levels, and atherosclerosis. Conclusions Our study identifies ARG2 as a key regulator of monocyte-mediated vascular inflammation and atherogenesis by controlling integrin, chemoattractant receptor expression, and pro-inflammatory cytokine release through TLR4-ERK-NFκB signaling, highlighting ARG2 as a potential therapeutic target for vascular and chronic inflammatory diseases.

Aging is a predominant risk factor for heart disease. Aging heart reveals low-grade chronic inflammation, cell apoptosis, cardiac fibrosis, and increased vulnerability to ischemic injury. The underlying molecular mechanisms responsible for cardiac aging and its susceptibility to injury are not fully understood. Although literature reports a role for mitochondrial Arginase 2 (ARG2) in heart failure, contradictory results are reported. How ARG2 participates in cardiac aging is still unknown. In this study, we demonstrate that Arg2 is not expressed in cardiomyocytes from aged mice and humans but is upregulated in non-myocyte cells, including macrophages, fibroblasts, and endothelial cells. Mice with genetic deficiency of Arg2 (Arg2-/-) are protected from age-associated cardiac inflammation, myocyte apoptosis, interstitial and perivascular fibrosis, endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic injury. Further experiments show that ARG2 mediates IL-1β release from macrophages of old mice, contributing to the cardiac aging phenotype. In addition, ARG2 enhances mitochondrial reactive oxygen species (mtROS) and activates cardiac fibroblasts that is inhibited by inhibition of mtROS. Thus, our study demonstrates a non-cell-autonomous effect of ARG2 on cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1β from aging macrophages as well as a cell-autonomous effect of ARG2 through mtROS in fibroblasts contributing to cardiac aging phenotype.

BackgroundAberrant mitochondrial metabolism is a key source of massive mitochondrial reactive oxygen species (mtROS) in tumour cells. Arginase-II (Arg-II), a widely expressed mitochondrial metabolic enzyme, has recently been shown to enhance mtROS production and melanoma progression. However, how Arg-II enhances mtROS and whether mtROS is involved in stimulation of cancer cell proliferation and migration remain unclear.Methods and resultsHere, we show that ablation of arg-ii suppresses cell growth, migration, nuclear deformation, and DNA damage in melanoma cells. Vice versa, overexpression of arg-ii in melanoma cells promotes melanoma cell growth and migration accompanied by enhanced nuclear deformation and DNA damage. Ablation or overexpression of arg-ii reduces or enhances mtROS, respectively, accounting for the effects of Arg-II on melanoma growth, migration, and DNA damage. Further data demonstrate that Arg-II enhances mtROS through decreasing Sirtuin 3 (Sirt3) levels. Silencing sirt3 promotes melanoma growth, migration, nuclear deformation, and DNA damage through enhancing mtROS. In supporting of these findings, overexpression of sirt3 prevented Arg-II-induced mtROS production with concomitant prevention of Arg-II-induced cell growth, migration, nuclear deformation and DNA damage. Furthermore, we show that upregulation of Arg-II under hypoxia induces nuclear deformation and DNA damage through suppressing Sirt3. Similar results are obtained in A549 human lung carcinoma cells. In addition, analysis of publicly accessible datasets reveals that elevated arg-ii mRNA levels in human tumor samples including skin cutaneous melanoma and lung cancers associate with poorer prognosis.ConclusionAltogether, our findings demonstrate a critical role of Arg-II-Sirt3-mtROS cascade in promoting melanoma growth, migration, nuclear deformation, and DNA damage linking to melanoma progression and malignancy, which could be therapeutic targets for cancers such as melanoma and lung carcinoma.

Aging is a predominant risk factor for cardiovascular diseases. There is evidence demonstrating that senescent cells not only play a significant role in organism aging but also contribute to the pathogenesis of cardiovascular diseases in younger ages. Encouraged by recent findings that the elimination of senescent cells by pharmacogenetic tools could slow down and even reverse organism aging in animal models, senolytic drugs have been developed, and the translation of results from basic research to clinical settings has been initiated. Because numerous studies in the literature show beneficial therapeutic effects of targeting senescent cells in cardiomyopathies associated with aging and ischemia/reperfusion and in atherosclerotic vascular disease, senolytic drugs are considered the next generation of therapies for cardiovascular disorders. However, recent studies have reported controversial results or detrimental effects caused by senolytic therapeutic approaches, including worsening of cardiac dysfunction, instability of atherosclerotic plaques, and even an increase in mortality in animal models, which challenges the translation of senolytic therapy into the clinical practice. This brief review article will focus on (1) analyzing and discussing the beneficial and detrimental effects of senolytic therapeutic approaches in cardiovascular diseases and cardiovascular aging and (2) future research directions and questions that are essential to understand the controversies and to translate preclinical results of senolytic therapies into clinical practice.

Age-associated sarcopenia decreases mobility and is promoted by cell senescence, inflammation, and fibrosis. The mitochondrial enzyme arginase-II (Arg-II) plays a causal role in aging and age-associated diseases. Therefore, we aim to explore the role of Arg-II in age-associated decline of physical activity and skeletal muscle aging in a mouse model. Young (4-6 months) and old (20-24 months) wild-type (wt) mice and mice deficient in arg-ii (arg-ii-/-) of both sexes are investigated. We demonstrate a decreased physical performance of old wt mice, which is partially prevented in arg-ii-/- animals, particularly in males. The improved phenotype of arg-ii-/- mice in aging is associated with reduced sarcopenia, cellular senescence, inflammation, and fibrosis, whereas age-associated decline of microvascular endothelial cell density, satellite cell numbers, and muscle fiber types in skeletal muscle is prevented in arg-ii-/- mice. Finally, we demonstrate an increased arg-ii gene expression level in aging skeletal muscle and found Arg-II protein expression in endothelial cells and fibroblasts, but not in skeletal muscle fibers, macrophages, and satellite cells. Our results suggest that increased Arg-II in non-skeletal muscle cells promotes age-associated sarcopenia, particularly in male mice.

Abstract Background Aging-associated cardiac structural and functional alterations enhance cardiac susceptibility to ischemic injury. The full elucidation of the underlying molecular mechanisms remains far from complete. The mitochondrial enzyme arginase-II (Arg-II) is implicated in cardiac injury and aging. However, contradictory results have been reported and no systemic analysis of its cellular localization has been performed. The involvement of Arg-II in cardiac aging, as well as the manner in which it participates, remains uncertain. Aim and methods: This project focuses on investigation of Arg-II cellular localization and its functional role in cardiac aging. Experiments are conducted in young (3-4 months) and old (20-22 months) wt and arg-ii-/- mice and in human heart tissues. Cardiac function is assessed by Langendorff apparatus ex vivo. Cellular localization of Arg-II in cardiac tissues is investigated by confocal microscopy. Intercellular interactions are studied using ventricular cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages isolated from wt and arg-ii-/- mice or humans. Results Cardiac Arg-II expression is enhanced with aging. Surprisingly, Arg-II is not found in cardiomyocytes from aged mice and human patients, but upregulated in non-myocytes, including macrophages, fibroblasts, endothelial cells. Arg-ii-/- mice are protected from age-associated cardiac inflammation, cardiomyocyte apoptosis, fibrosis, endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic injury. Further experiments show that Arg-II mediates IL-1b release from macrophages of old mice, contributing to the above-described cardiac aging phenotype. In addition, Arg-II enhances mitochondrial reactive oxygen species (mtROS) and activates cardiac fibroblasts that is inhibited by inhibition of mtROS. Conclusion Our study demonstrates a non-cell-autonomous effect of Arg-II on cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages, which may explain the enhanced vulnerability of aging heart to ischemic injury.Role of Arg-II in cardiac aging

Increased endothelial permeability plays an important role in blood-brain barrier (BBB) dysfunction and is implicated in neuronal injury in many diseased conditions. BBB disruption is primarily determined by dysfunction of endothelial cell-cell junctions. Deprivation of oxygen supply or hypoxia, a common feature of a variety of human diseases, is a major risk factor for BBB disruption. The molecular regulatory mechanisms of hypoxiainduced BBB dysfunction remain incompletely understood. The mitochondrial enzyme, arginase type II (Arg-II), has been shown to promote endothelial dysfunction. However, its role in hypoxia-induced BBB dysfunction has not been explored. In the C57BL/6J mouse model, hypoxia (8% O2, 24 hours) augments vascular Arg-II in the hippocampus, decreases cell-cell junction protein levels of Zonula occludens-1 (ZO-1), occludin, and CD31 in endothelial cells, increases BBB leakage in the brain in old mice (20 to 24 months) but not in young animals (3 to 6 months). These effects of hypoxia in aging are suppressed in arg-ii-/-mice. Moreover, the age-associated vulnerability of endothelial integrity to hypoxia is demonstrated in senescent human brain microvascular endothelial cell (hCMEC/D3) culture model. Further results in the cell culture model show that hypoxia augments Arg-II, decreases ZO-1 and occludin levels, and increases endothelial permeability, which is prevented by arg-ii gene silencing or by inhibition of mitochondrial reactive oxygen species (mtROS) production. Our study demonstrates an essential role of Arg-II in increased endothelial permeability and BBB dysfunction by promoting mtROS generation, resulting in decreased endothelial cell-cell junction protein levels under hypoxic conditions particularly in aging.

Elevated arginases including type‐I (Arg‐I) and type‐II isoenzyme (Arg‐II) are reported to play a role in aging, age‐associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg‐II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg‐II is also observed in human lung biopsies. The age‐associated increase in lung fibrosis and inflammatory cytokines, including IL‐1β and TGF‐β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg‐ii deficient ( arg‐ii −/− ) mice. The effects of arg‐ii − /− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg‐II‐positive bronchial and alveolar epithelial cells, but not that from arg‐ii −/− cells, activates fibroblasts to produce various cytokines including TGF‐β1 and collagen, which is abolished by IL‐1β receptor antagonist or TGF‐β type I receptor blocker. Conversely, TGF‐β1 or IL‐1β also increases Arg‐II expression. In the mouse models, we confirmed the age‐associated increase in IL‐1β and TGF‐β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg‐ii − /− mice. Taken together, our study demonstrates a critical role of epithelial Arg‐II in activation of pulmonary fibroblasts via paracrine release of IL‐1β and TGF‐β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg‐II in pulmonary aging.

One of the manifestations of renal aging is podocyte dysfunction and loss, which are associated with proteinuria and glomerulosclerosis. Studies show a male bias in glomerular dysfunction and chronic kidney diseases, and the underlying mechanisms remain obscure. Recent studies demonstrate the role of an age-associated increase in arginase-II (Arg-II) in proximal tubules of both male and female mice. However, it is unclear whether Arg-II is also involved in aging glomeruli. The current study investigates the role of the sex-specific elevation of Arg-II in podocytes in age-associated increased albuminuria. Young (3-4 months) and old (20-22 months) male and female mice of wt and arginase-II knockout (arg-ii(-/-)) were used. Albuminuria was employed as a readout of glomerular function. Cellular localization and expression of Arg-II in glomeruli were analyzed using an immunofluorescence confocal microscope. A more pronounced age-associated increase in albuminuria was found in male than in female mice. An age-associated induction of Arg-II in glomeruli and podocytes (as demonstrated by co-localization of Arg-II with the podocyte marker synaptopodin) was also observed in males but not in females. Ablation of the arg-ii gene in mice significantly reduces age-associated albuminuria in males. Also, age-associated decreases in podocyte density and glomerulus hypertrophy are significantly prevented in male arg-ii(-/-) but not in female mice. However, age-associated glomerulosclerosis is not affected by arg-ii ablation in both sexes. These results demonstrate a role of Arg-II in sex-specific podocyte injury in aging. They may explain the sex-specific differences in the development of renal disease in humans during aging.

Hypoxia is an important risk for renal disease. The mitochondrial enzyme arginase-II (Arg-II) is expressed and/or induced by hypoxia in proximal tubular epithelial cells (PTECs) and in podocytes, leading to cellular damage. Because PTECs are vulnerable to hypoxia and located in proximity to podocytes, we examined the role of Arg-II in the crosstalk of PTECs under hypoxic conditions with podocytes. A human PTEC cell line (HK2) and a human podocyte cell line (AB8/13) were cultured. Arg-ii gene was ablated by CRISPR/Case9 in both cell types. HK2 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 48 h. Conditioned medium (CM) was collected and transferred to the podocytes. Podocyte injuries were then analyzed. Hypoxic (not normoxic) HK2-CM caused cytoskeletal derangement, cell apoptosis, and increased Arg-II levels in differentiated podocytes. These effects were absent when arg-ii in HK2 was ablated. The detrimental effects of the hypoxic HK2-CM were prevented by TGF-β1 type-I receptor blocker SB431542. Indeed, TGF-β1 levels in hypoxic HK2-CM (but not arg-ii−/−-HK2-CM) were increased. Furthermore, the detrimental effects of TGF-β1 on podocytes were prevented in arg-ii−/−-podocytes. This study demonstrates crosstalk between PTECs and podocytes through the Arg-II-TGF-β1 cascade, which may contribute to hypoxia-induced podocyte damage.

Hypoxia plays a crucial role in acute and chronic renal injury, which is attributable to renal tubular and glomerular cell damage. Some studies provide evidence that hypoxia-dependent upregulation of the mitochondrial enzyme arginase type-II (Arg-II) in tubular cells promotes renal tubular injury. It is, however, not known whether Arg-II is also expressed in glomerular cells, particularly podocytes under hypoxic conditions, contributing to hypoxia-induced podocyte injury. The effects of hypoxia on human podocyte cells (AB8/13) in cultures and on isolated kidneys from wild-type (wt) and arg-ii gene-deficient (arg-ii−/−) mice ex vivo, as well as on mice of the two genotypes in vivo, were investigated, respectively. We found that the Arg-II levels were enhanced in cultured podocytes in a time-dependent manner over 48 h, which was dependent on the stabilization of hypoxia-inducible factor 1α (HIF1α). Moreover, a hypoxia-induced derangement of cellular actin cytoskeletal fibers, a decrease in podocin, and an increase in mitochondrial ROS (mtROS) generation—as measured by MitoSOX—were inhibited by adenoviral-mediated arg-ii gene silencing. These effects of hypoxia on podocyte injury were mimicked by the HIFα stabilizing drug DMOG, which inhibits prolyl hydroxylases (PHD), the enzymes involved in HIFα degradation. The silencing of arg-ii prevented the detrimental effects of DMOG on podocytes. Furthermore, the inhibition of mtROS generation by rotenone—the inhibitor of respiration chain complex-I—recapitulated the protective effects of arg-ii silencing on podocytes under hypoxic conditions. Moreover, the ex vivo experiments with isolated kidney tissues and the in vivo experiments with mice exposed to hypoxic conditions showed increased Arg-II levels in podocytes and decreased podocyte markers regarding synaptopodin in wt mice but not in arg-ii−/− mice. While age-associated albuminuria was reduced in the arg-ii−/− mice, the hypoxia-induced increase in albuminuria was, however, not significantly affected in the arg-ii−/−. Our study demonstrates that Arg-II in podocytes promotes cell injury. Arg-ii ablation seems insufficient to protect mice in vivo against a hypoxia-induced increase in albuminuria, but it does reduce albuminuria in aging.

The ureohydrolase, type-II arginase (Arg-II), is a mitochondrial enzyme metabolizing L-arginine into urea and L-ornithine and is highly expressed in renal proximal tubular cells (PTC) and upregulated by renal ischemia. Recent studies reported contradictory results on the role of Arg-II in renal injury. The aim of our study is to investigate the function of Arg-II in renal epithelial cell damage under hypoxic conditions. Human renal epithelial cell line HK2 was cultured under hypoxic conditions for 12–48 h. Moreover, ex vivo experiments with isolated kidneys from wild-type (WT) and genetic Arg-II deficient mice (Arg-II–/–) were conducted under normoxic and hypoxic conditions. The results show that hypoxia upregulates Arg-II expression in HK2 cells, which is inhibited by silencing both hypoxia-inducible factors (HIFs) HIF1α and HIF2α. Treatment of the cells with dimethyloxaloylglycine (DMOG) to stabilize HIFα also enhances Arg-II. Interestingly, hypoxia or DMOG upregulates transforming growth factor β1 (TGFβ1) levels and collagens Iα1, which is prevented by Arg-II silencing, while TGFβ1-induced collagen Iα1 expression is not affected by Arg-II silencing. Inhibition of mitochondrial complex-I by rotenone abolishes hypoxia-induced reactive oxygen species (mtROS) and TGFβ1 elevation in the cells. Ex vivo experiments show elevated Arg-II and TGFβ1 expression and the injury marker NGAL in the WT mouse kidneys under hypoxic conditions, which is prevented in the Arg-II–/– mice. Taking together, the results demonstrate that hypoxia activates renal epithelial HIFs-Arg-II-mtROS-TGFβ1-cascade, participating in hypoxia-associated renal injury and fibrosis.

The aging kidney undergoes complex changes and is vulnerable to injury and development of chronic kidney disease (CKD) with preponderance affecting more women than men. Evidence has been presented that the type-II L-arginine:ureohydrolase, arginase-II (Arg-II) plays a role in the acceleration of aging. Arg-II is highly expressed in the kidney. However, the role of Arg-II in renal aging is not known. This study is to investigate whether Arg-II is involved in the kidney aging process dependently on sex. Arg-II level in the kidney of wild type (WT) mice is significantly elevated with aging, which is accompanied by an increase in expression of the inflammatory cytokines/chemokines, tissue macrophages, factors involved in fibrosis, and tubulointestitial fibrosis in both males and females. This renal aging phenotype is significantly suppressed in arg-II −/− mice, mainly in the females in which Arg-II level is higher than in the males. Importantly, numerous factors such as IL-1β, MCP1, VCAM-1, and TGFβ1 are mainly localized in the proximal tubular S3 segment cells expressing Arg-II in the aging kidney. In human proximal tubular cells (HK-2), TNF-α enhances adhesion molecule expression dependently on Arg-II upregulation. Overexpression of Arg-II in the cells enhances TGFβ1 levels which is prevented by mitochondrial ROS inhibition. In summary, our study reveals that renal proximal tubular Arg-II plays an important role in the kidney aging process in females. Arg-II could be a promising therapeutic target for the treatment and prevention of aging-associated kidney diseases.

The impaired L-arginine/nitric oxide pathway is a well-recognized mechanism for cardiovascular and renal diseases with aging. Therefore, supplementation of L-arginine is widely proposed to boost health or as adjunct therapy for the patients. However, clinical data, show adverse effects and even enhanced mortality in patients receiving long-term L-arginine supplementation. The effects of long-term L-arginine supplementation on kidney aging and the underlying mechanisms remain elusive. Moreover, high protein and high amino acid diet has been thought detrimental for kidney. We therefore investigated effects of chronic dietary L-arginine supplementation on kidney aging. In both young (4 months) and old (18–24 months) mice, animals either receive standard chow containing 0.65% L-arginine or diet supplemented with L-arginine to 2.46% for 16 weeks. Inflammation and fibrosis markers and albuminuria are then analyzed. Age-associated increases in tnf-α , il-1β , and il-6 , vcam-1 , icam-1 , mcp1 , inos , and macrophage infiltration, collagen expression, and S6K1 activation are observed, which is not favorably affected, but rather further enhanced, by L-arginine supplementation. Importantly, L-arginine supplementation further enhances age-associated albuminuria and mortality particularly in females, accompanied by elevated renal arginase-II (Arg-II) levels. The enhanced albuminuria by L-arginine supplementation in aging is not protected in Arg-II −/− mice. In contrast, L-arginine supplementation increases ROS and decreases nitric oxide production in old mouse aortas, which is reduced in Arg-II −/− mice. The results do not support benefits of long-term L-arginine supplementation. It rather accelerates functional decline of kidney and vasculature in aging. Thus, the long-term dietary L-arginine supplementation should be avoided particularly in elderly population.

Elevated arginase type II (Arg-II) associates with higher grade tumors. Its function and underlying molecular mechanisms in melanoma remain elusive. In the present study, we observed a significantly higher frequency of Arg-II expression in melanoma of patients with metastasis than those without metastasis. Silencing Arg-II in two human melanoma cell lines slowed down the cell growth, while overexpression of native but not a catalytically inactive Arg-II promoted cell proliferation without affecting cell death. Treatment of cells with arginase inhibitor also reduced melanoma cell number, demonstrating that Arg-II promotes melanoma cell proliferation dependently of its enzymatic activity. However, results from silencing Arg-II or overexpressing native or the inactive Arg-II as well as treatment with arginase inhibitor showed that Arg-II promotes melanoma metastasis-related processes, such as melanoma cell migration and adhesion on endothelial cells, independently of its enzymatic activity. Moreover, the treatment of the cells with STAT3 inhibitor suppressed Arg-II-promoted melanoma cell migration and adhesion. Furthermore, catalase, but not superoxide dismutase, prevented STAT3 activation as well as increased melanoma cell migration and adhesion induced by overexpressing native or the inactive Arg-II. Taken together, our study uncovers both activity-dependent and independent mechanisms of Arg-II in promoting melanoma progression. While Arg-II enhances melanoma cell proliferation through polyamine dependently of its enzymatic activity, it promotes metastasis-related processes, that is, migration and adhesion onto endothelial cell, through mitochondrial H2 O2 -STAT3 pathway independently of the enzymatic activity. Suppressing Arg-II expression rather than inhibiting its enzymatic activity may, therefore, represent a novel strategy for the treatment of melanoma.

Background: Adipose tissue inflammation occurs not only in obesity but also in aging and is mechanistically linked with age-associated diseases. Studies show that ablation of the l-arginine-metabolizing enzyme arginase-II (Arg-II) reduces adipose tissue inflammation and improves glucose tolerance in obesity. However, the role of Arg-II in aging adipose tissue inflammation is not clear. Objective: This study investigated the role of Arg-II in age-associated adipose tissue inflammation. Methods: Visceral adipose tissues of young (3–6 months) and old (20–24 months) wild-type (WT) and Arg-II−/− mice were investigated. Immunofluorescence confocal microscopy was performed for analysis of macrophage accumulation and cellular localization of arginase and cytokines; expression of arginase and cytokines was analyzed by qRT-PCR or immunoblotting or ELISA; activation of mitogen-activated protein kinases in adipose tissues was analyzed by immunoblotting; and arginase activity was measured by colorimetric determination of urea production. Results: In the old WT mice, there is more macrophage accumulation in the visceral adipose tissues than in Arg-II knockout animals. An age-associated increase in arginase activity and Arg-II expression in adipose tissues of WT mice is observed. Arg-II knockout enhances Arg-I expression and activity, but inhibits interleukin (IL)-6 expression and secretion and reduces active p38mapk in aging adipose tissue macrophages and stromal cells. Treatment of aging adipose tissues of WT mice with a specific p38mapk inhibitor SB203580 reduces IL-6 secretion. Conclusions: Arg-II promotes IL-6 production in aging adipose tissues through p38mapk. The results suggest that targeting Arg-II or inhibiting p38mapk could be beneficial in reducing age-associated adipose tissue inflammation.

Insulin-induced AKT activation is dependent on phosphoinositide 3-kinase and opposed by tumor suppressor phosphatase and tensin homolog (PTEN). Our previous study demonstrates that myosin 1b (MYO1B) mediates arginase-II-induced activation of mechanistic target of rapamycin complex 1 that is regulated by AKT. However, the role of MYO1B in AKT activation is unknown. Here we show that silencing MYO1B in mouse embryonic fibroblasts (MEF) inhibits insulin-induced nuclear but not cytoplasmic AKT activation accompanied by elevated nuclear PTEN level. Co-immunoprecipitation, co-immunostaining, and proximity ligation assay show an interaction of MYO1B and PTEN resulting in reduced nuclear PTEN. Moreover, the elevated nuclear PTEN upon silencing MYO1B promotes apoptosis of MEFs and melanoma B16F10 cells. Taken together, we demonstrate that MYO1B, by interacting with PTEN, prevents nuclear localization of PTEN contributing to nuclear AKT activation and suppression of cell apoptosis. This may present a therapeutic approach for cancer treatment such as melanoma.