Yeong-Min Yoo

Melatonin suppresses cyclosporine A-induced autophagy in rat pituitary GH3 cells

Abstract:  Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0–100 μm) decreased cell survival of rat pituitary GH3 cells in a dose‐dependent manner. At concentrations ranging from 1.0 to 10 μm, CsA induced a dose‐dependent increase in the expression of microtubule‐associated protein 1 light chain 3 (LC3)‐I and LC3‐II. Cells treated with 2.5 μm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0–10 μm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol‐requiring enzyme 1 alpha (IRE1α), increased as compared those levels in untreated cells. These results suggested that CsA‐induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA‐induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 μm) suppressed autophagy induced by 2.5 and 5 μm CsA. Furthermore, co‐treatment with 100 μm melatonin inhibited LC3‐II expression, and increased catalase and phosphorylated p‐ERK levels in the presence of 2.5 and 5 μm CsA. BiP and IRE1α expression in melatonin‐co‐treated cells was superior to that in cells treated with 2.5 and 5 μm CsA alone. Thus, melatonin suppresses CsA‐mediated autophagy in rat pituitary GH3 cells.

Melatonin protects against apoptotic and autophagic cell death in C2C12 murine myoblast cells.

Abstract:  In this study, we investigated whether or not melatonin inhibits apoptotic and autophagic cell death in C2C12 murine myoblast cells. Treatment of cells with S‐nitroso‐N‐acetylpenicillamine (SNAP), an NO donor, was shown to induce cell death, and treatment with melatonin (100 μm) significantly attenuated the occurrence of NO‐induced cell death. Decreased p‐Akt expression in response to NO was also arrested by melatonin. Under these conditions, p‐Bad (Ser 136) expression increased with melatonin treatment prior to NO treatment. Treatment with Akt inhibitors (LY 294002, wortmannin) plus melatonin reduced p‐Akt expression. Compared with NO treatment, Bcl‐2 expression increased with melatonin treatment, while Bax expression was inhibited by melatonin treatment. Expression of catalase and Mn‐superoxide dismutase (SOD) was elevated with melatonin treatment, whereas Cu/Zn‐SOD expression decreased with melatonin, lower than NO treatment, respectively. Next, we investigated the question of whether or not melatonin may restrain autophagic cell death in C2C12 cells. Nutrient starvation induced a rise in expression of the microtubule‐associated protein 1 light chain 3 (LC3)‐II; however, melatonin treatment suppressed LC3‐II expression by nutrient deprivation. Expression of Bcl‐2, Bax, catalase, and Cu/Zn‐SODs coincided with results of apoptotic cell death. Together, these results suggest that melatonin protects against apoptotic and autophagic cell death through the common pathway resulted in the increment of Bcl‐2 expression and the reduction of Bax expression in C2C12 murine myoblast cells.

Effects of Neuropeptides and Mechanical Loading on Bone Cell Resorption in Vitro

Neuropeptides such as vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are present in nerve fibers of bone tissues and have been suggested to potentially regulate bone remodeling. Oscillatory fluid flow (OFF)-induced shear stress is a potent signal in mechanotransduction that is capable of regulating both anabolic and catabolic bone remodeling. However, the interaction between neuropeptides and mechanical induction in bone remodeling is poorly understood. In this study, we attempted to quantify the effects of combined neuropeptides and mechanical stimuli on mRNA and protein expression related to bone resorption. Neuropeptides (VIP or CGRP) and/or OFF-induced shear stress were applied to MC3T3-E1 pre-osteoblastic cells and changes in receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) mRNA and protein levels were quantified. Neuropeptides and OFF-induced shear stress similarly decreased RANKL and increased OPG levels compared to control. Changes were not further enhanced with combined neuropeptides and OFF-induced shear stress. These results suggest that neuropeptides CGRP and VIP have an important role in suppressing bone resorptive activities through RANKL/OPG pathway, similar to mechanical loading.

Effect of melatonin on mRNA expressions of transcription factors in murine embryonic stem cells.

Murine embryonic stem (ES) cells constitute a versatile biological system that has facilitated major advances in the fields of cell and developmental biology. Although melatonin has a wide variety of biological functions, its effect on ES cells is still unknown. In this study, we examined the effects of melatonin on transcription factors of ES cells (ES-E14TG2a cells) using an in vitro culture system. We found that melatonin affected cellular proliferation and Akt phosphorylation. Melatonin treatment also increased Bcl-2 expression and suppressed Bax expression and decreased phosphorylation of GSK α/β. The transcription factor Oct4, which contains the POU (N-terminal to homeobox) domain, and the transcription factor Sox2, the zinc finger transcription factor Zfp206, and the zinc finger gene REX-1 (Znf42), which contain the high mobility group (HMG) domain, are all important for cellular pluripotency and pre-implantation development. In this study, melatonin treatment influenced Oct4 and REX-1 expression at day 1 but not significantly at days 2 and 3. In addition, Sox2 and Zfp206 expressions did not change over the course of melatonin treatment. Taken together, melatonin may affect Akt activation but does not influence the mRNA expressions of Oct4, REX-1, Sox2, or Zfp206, suggesting that melatonin may stimulate proliferation of ES-E14TG2a cells via Akt phosphorylation.

Melatonin‐mediated insulin synthesis during endoplasmic reticulum stress involves HuD expression in rat insulinoma INS‐1E cells

In this study, we investigated how melatonin mediates insulin synthesis through endoplasmic reticulum (ER) via HuD expression in rat insulinoma INS‐1E cells. Under ER stress condition (thapsigargin with/without melatonin, tunicamycin with/without melatonin), phosphorylation of AMP‐activated protein kinase (p‐AMPK) was significantly increased when compared with only with/without melatonin (control/melatonin). Insulin receptor substrate (IRS) two protein was significantly reduced under conditions of ER stress when compared with control/melatonin, but no expression of IRS1 protein was observed. In thapsigargin treatment, melatonin (10, 50 μm) increased IRS2 protein expression in a dose‐dependent manner. p‐Akt (Ser473) expression significantly decreased under ER stress condition prior to control/melatonin. Melatonin (10, 50 μm) significantly reduced nuclear and cellular p85α expressions in a dose‐dependent manner when compared with only thapsigargin or tunicamycin. These results indicate the activation of the aforementioned expressions under regulation of the pathway, AMPK → IRS2 → Akt/PKB → PI3K (p85α). However, mammalian target of rapamycin and raptor protein, mTORC1, was found to be independent of the ER stress response. In thapsigargin treatment, melatonin increased nuclear mammalian RNA‐binding protein (HuD) expression and reduced cellular HuD expression and subsequently resulted in a decrease in cellular insulin level and rise in insulin secretion in a dose‐dependent manner. In tunicamycin treatment, HuD and insulin proteins showed similar expression tendencies. These results indicate that ER stress/melatonin, especially thapsigargin/melatonin, increased nuclear HuD expression and subsequently resulted in a decrease in intracellular biosynthesis; it is hypothesized that extracellular secretion of insulin may be regulated by melatonin.

Melatonin-induced autophagy is associated with degradation of MyoD protein in C2C12 myoblast cells

Abstract:  MyoD is a muscle‐specific transcriptional factor that acts as a master switch for skeletal muscle differentiation. This protein regulates myoblast proliferation and myogenic differentiation and is also a short‐lived regulatory protein that is degraded by the ubiquitin system. However, the lysosomal pathway of MyoD protein degradation remains unknown. In this study, we sought to determine whether melatonin (1, 2 mm)‐induced autophagy causes the degradation of MyoD protein in C2C12 myoblast cells. Melatonin induced a significant increase in expression of the microtubule‐associated protein 1 light chain 3 (LC3)‐II and Beclin‐1 proteins in a dose‐dependent manner. Melatonin treatment also significantly increased p‐ERK, Ras, and p‐Akt expressions in a dose‐dependent manner. However, Bax expression was high compared with the absence of melatonin treatment, and Bcl‐2 expression was high in the 0.1–0.5 mm melatonin treatments and low in the 1 and 2 mm melatonin treatments. Under the same conditions, cytosolic MyoD protein was significantly decreased in a dose‐dependent manner and completely eliminated by 36 hr. This decrease in MyoD protein involved ubiquitin‐mediated proteasomal activity with proteasome inhibitor MG132 or autophagy‐dependent lysosomal degradation with lysosomal inhibitor bafilomycin A1 (Baf‐A1). In the same condition, phosphorylation of the mammalian target of rapamycin, p‐mTOR, and p‐S6K expression with Baf‐A1 or Baf‐A1‐plus melatonin treatment were significantly decreased compared with the levels after treatment with melatonin only. Together, these results suggest that melatonin (1, 2 mm)‐induced autophagy results in partial lysosomal degradation of MyoD protein in C2C12 myoblast cells.

Cyclosporine A induces apoptotic and autophagic cell death in rat pituitary GH3 cells.

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.

Fluid shear stress and melatonin in combination activate anabolic proteins in MC3T3‐E1 osteoblast cells

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal‐regulated kinase (p‐ERK) in MC3T3‐E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1 hr) and melatonin (1 mm) in combination reduced autophagic marker LC3‐II compared with fluid shear stress (1 hr) and/or melatonin (0.1 mm). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p‐ERK, phosphorylation of serine‐threonine protein kinase (p‐Akt), phosphorylation of mammalian target of rapamycin (p‐mTOR), and p85‐S6K were investigated. p‐Akt, p‐mTOR (Ser 2481) expressions increased with the addition of 1 mm melatonin prior to 0.1 mm melatonin treatment. However, p‐S6K expression did not change significantly. Next, mitochondria activity including Bcl‐2, Bax, catalase, and Mn‐superoxide dismutase (Mn‐SOD) were studied. Expressions of Bcl‐2, Bax, and catalase proteins were low under fluid shear stress plus 1 mm melatonin compared with only fluid shear stress alone, whereas Mn‐SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4 hr) and melatonin (0.1, 1 mm). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p‐ERK in MC3T3‐E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts.

Parathyroid hormone-related protein and glucocorticoid receptor beta are regulated by cortisol in the kidney of male mice

AIMS Parathyroid hormone-related protein (PTHrP) is a peptide growth factor produced in a wide range of tissues from brain and parathyroid, to kidney and uterus. The purpose of this study was to determine whether the adrenal cortical hormones, hydrocortisone (cortisol), modulate PTHrP expression and glucocorticoid receptor (GR)β in mice kidney. MAIN METHODS Changes in PTHrP gene expression were determined by real-time PCR and its protein level was examined by Western blot analysis. In addition, expression of renal PTHrP protein was localized by immunohistochemistry. Effects of RU486 on the expression levels of GRα/β or PTHrP gene in the kidneys were analyzed by Western blot analysis. KEY FINDINGS We found that renal expression levels of PTHrP mRNA were higher in males than in females up to 9weeks of age. Using immunohistochemistry, we observed higher levels of PTHrP expression within the cortex than in the medulla in both male and female mice, and this expression was localized in the epithelial cells of the renal proximal tubules. Treatment of 4-week-old mice with aldosterone and cortisol for three days showed larger increases in both PTHrP mRNA and protein levels in males compared with females. The expression of GRβ in male, but not female, kidneys was significantly upregulated after treatment with cortisol, but not after treatment with aldosterone. Inhibition of glucocorticoid signaling by pre-treatment with a GR antagonist prior to cortisol administration largely abolished this cortisol-dependent increase in PTHrP and GRβ expressions. SIGNIFICANCE These results suggest that PTHrP expression and GRβ in the kidneys of male mice may be regulated by cortisol.

Pharmacological advantages of melatonin in immunosenescence by improving activity of T lymphocytes

Abstract Melatonin plays a critical role in regulating photoperiodic signals and has recently been shown to decrease immunosenescence with age. In this study, we examined whether melatonin activates T lymphocytes as major adaptive immune cells in in vitro and in vivo models. Splenocytes, CD4+, and naïve CD4 T lymphocytes were isolated from the spleen of BALB/c mice and the cell population patterns and mRNA profiles associated with T cell activation (CD28 and p21) and the melatonin receptor (MT1A and MT1B) were assessed. The T cell activation-related proteins Ki67 and Bcl2 were also evaluated to confirm the relationship between gene and protein levels. Our data clearly revealed that CD28, p21, MT1A, and MT1B mRNA were highly expressed in the presence of melatonin. Co-culture of CD4+ T lymphocyte and peritoneal macrophage 7 days after melatonin administration to young and aged mice significantly increased APRIL mRNA, suggesting induction or maintenance of T lymphocyte responses. We also found that the intracellular amount of Ki67 and Bcl2 proteins were significantly upregulated in aged CD4+ T lymphocytes, suggesting enhancing T cell proliferation and ling-term maintenance of memory T cells. Taken together, we conclude that melatonin supplementation may enhance immunity in aged individuals by upregulating immunosenescence indices in association with T lymphocytes and may be an attractive pharmacological candidate for aged and immunocompromised individuals.