Risk factors for disease progression in Japanese patients with COVID-19 with no or mild symptoms on admission.

BACKGROUND: Although the risk factors for coronavirus disease 2019 (COVID-19) mortality have been identified, there is limited information about the risk factors for disease progression after hospitalization among Japanese patients with COVID-19 exhibiting no or mild symptoms. METHODS: All 302 consecutive patients who were admitted to our institutions and diagnosed with COVID-19 between March and December 2020 were retrospectively assessed. Ultimately, 210 adult patients exhibiting no or mild symptoms on admission were included in the analysis. They were categorized into the stable (no oxygen needed) and worsened (oxygen needed) groups, and their characteristics and laboratory data were compared. RESULTS: Among 210 patients, 49 progressed to a severe disease stage, whereas 161 did not. The mean patient age was 52.14 years, and 126 (60.0%) patients were male. The mean body mass index (BMI) was 23.0 kg/m2, and 71 patients were overweight (BMI ≥ 25 kg/m2). Multivariate logistic analysis showed that old age, overweight, diabetes mellitus (DM), and high serum ferritin levels were independent risk factors for disease progression. CONCLUSIONS: Clinicians should closely observe patients with COVID-19, especially those with risk factors such as old age, overweight, DM, and high serum ferritin levels, regardless of whether they have no or mild symptoms.

The Attenuated Secretion of Hyaluronan by UVA-Exposed Human Fibroblasts Is Associated with Up- and Downregulation of HYBID and HAS2 Expression via Activated and Inactivated Signaling of the p38/ATF2 and JAK2/STAT3 Cascades.

Little is known about the effects on hyaluronan (HA) metabolism of UVA radiation. This study demonstrates that the secretion of HA by human dermal fibroblasts (HDFs) is downregulated by UVA, accompanied by the down- and upregulation of mRNA and protein levels of the HA-synthesizing enzyme (HAS2) and the HA-degrading protein, HYaluronan Binding protein Involved in HA Depolymerization(HYBID), respectively. Signaling analysis revealed that the exposure distinctly elicits activation of the p38/MSK1/CREB/c-Fos/AP-1 axis, the JNK/c-Jun axis, and the p38/ATF-2 axis, but downregulates the phosphorylation of NF-kB and JAK/STAT3. A signal inhibition study demonstrated that the inhibition of p38 significantly abrogates the UVA-accentuated mRNA level of HYBID. Furthermore, the inhibition of STAT3 significantly downregulates the level of HAS2 mRNA in non-UVA exposed HDFs. Analysis using siRNAs demonstrated that transfection of ATF-2 siRNA but not c-Fos siRNA abrogates the increased protein level of HYBID in UVA-exposed HDFs. An inhibitor of protein tyrosine phosphatase but not of protein serine/threonine phosphatase restored the diminished phosphorylation level of STAT3 at Tyr 705, accompanied by a significant abolishing effect on the decreased mRNA expression level of HAS2. Silencing with a protein tyrosine phosphatase PTP-Meg2 siRNA revealed that it abrogates the decreased phosphorylation of STAT3 at Tyr 705 in UVA-exposed HDFs. These findings suggest that the UVA-induced decrease in HA secretion by HDFs is attributable to the down- and upregulation of HAS2 and HYBID expression, respectively, changes that are mainly ascribed to the inactivated signaling of the STAT3 axis due to the activated tyrosine protein phosphatase PTP-Meg2 and the activated signaling of the p38/ATF2 axis, respectively.

Treatment with radiosynoviorthesis in hemophilic patients with and without inhibitor.

BACKGROUND: Spontaneous bleedings occurring into joints (hemarthrosis) are the most common manifestations of hemophilia and causes severe joint damage ultimately resulting in joint disfunction known as hemophilic arthropathy. Among available therapeutic options for reducing recurrent hemarthrosis-associated damage, radiosynoviorthesis (RS) has proven effective in improving joint function. AIM: To assess the impact of RS with Yttrium(90) citrate (C-Y(90)) on frequency of hemarthroses and joint function in a group of pediatric patients. METHODS: Between November 1998 and February 2017, we evaluated 27 pediatric patients with mild, moderate or severe hemophilia with haemophilic arthropathy. Overall, RS was applied in 60 joints. Some patients received more than one single intra-articular injection with C-Y(90). RESULTS: During the follow-up, one patient showed joint bleeding 15 months after RS, one patient after 12 months and one patient after 45 days. The episodes of hemarthrosis were reduced and joint function significantly improved in all patients. CONCLUSION: RS with C-Y(90) is a simple and safe treatment for reducing the frequency of hemarthroses in patients with hemophilia. It decreases the use of factor VIII / IX and improves joint function.

Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

Apobec2 is a member of the activation-induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms of myopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z-lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2-deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2-/- mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10-mo-old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.-Sato, Y., Ohtsubo, H., Nihei, N., Kaneko, T., Sato, Y., Adachi, S.-I., Kondo, S., Nakamura, M., Mizunoya, W., Iida, H., Tatsumi, R., Rada, C., Yoshizawa, F. Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle.

Skeletal muscle-derived exosomes regulate endothelial cell functions via reactive oxygen species-activated nuclear factor-κB signalling.

NEW FINDINGS: What is the central question of this study? Capillary rarefaction is found in diabetic and aged muscle, whereas exercise increases skeletal muscle angiogenesis. The association implies a crosstalk between muscle cells and endothelial cells. The underlying mechanisms mediating the crosstalk between these cells remains to be elucidated fully. What is the main finding and its importance? Endothelial cell functions are regulated by skeletal muscle cell-derived exosomes via a vascular endothelial growth factor-independent pathway. This study reveals a new mechanism mediating the crosstalk between skeletal muscle cells and endothelial cells. ABSTRACT: Loss of skeletal muscle capillarization, known as capillary rarefaction, is found in type 2 diabetes, chronic heart failure and healthy ageing and is associated with impaired delivery of substrates to the muscle. However, the interaction and communication of skeletal muscle with endothelial cells in the regulation of capillaries surrounding the muscle remains elusive. Exosomes are a type of secreted extracellular vesicle containing mRNAs, proteins and, especially, microRNAs that exert paracrine and endocrine effects. In this study, we investigated whether skeletal muscle-derived exosomes (SkM-Exo) regulate the endothelial cell functions of angiogenesis. We demonstrated that C2C12 myotube-derived exosomes improved endothelial cell functions, assessed by the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), which were increased by 20, 23 and 40%, respectively, after SkM-Exo exposure. The SkM-Exo failed to activate HUVEC vascular endothelial growth factor (VEGF) signalling. The SkM-Exo increased HUVEC reactive oxygen species and activated the nuclear factor-κB pathway, suggesting that SkM-Exo-induced angiogenesis was mediated by a VEGF-independent pathway. In addition, several angiogenic microRNAs were packaged in SkM-Exo, with miR-130a being particularly enriched and successfully transferred from SkM-Exo to HUVECs. Delivery of miRNAs into endothelial cells might explain the enhancement of reactive oxygen species production and angiogenesis by SkM-Exo. The potential angiogenic effect of SkM-Exo could provide an effective therapy for promoting skeletal muscle angiogenesis in diseases characterized by capillary rarefaction or inadequate angiogenesis.

Leucyl-tRNA synthetase is required for the myogenic differentiation of C2C12 myoblasts, but not for hypertrophy or metabolic alteration of myotubes.

Mammalian target of rapamycin (mTOR) signaling controls skeletal muscle cell differentiation, growth, and metabolism by sensing the intracellular energy status and nutrients. Recently, leucyl-tRNA synthetase (Lars) was identified as an intracellular sensor of leucine involved in the activation of mTOR signaling. However, there is still no evidence for the activation of mTOR signaling by Lars and its physiological roles in skeletal muscle cells. In this study, we determined the potential roles of Lars for the activation of mTOR signaling, skeletal muscle cell differentiation, hypertrophy, and metabolism using small interfering (si)-RNA knockdown. siRNA-mediated knockdown of Lars decreased phosphorylated p70 S6 kinase and inhibited the differentiation of C2C12 mouse myoblasts into myotubes, as evidenced by a decreased fusion index and decreased mRNA and protein expression levels of myogenic markers. Importantly, si-Lars decreased the level of Insulin-like growth factor 2 (Igf2) mRNA expression from the early stages of differentiation, indicating the possibility of an association between the mTOR-IGF2 axis and Lars. However, Lars knockdown did not decrease phosphorylated mTOR in differentiated myotubes, nor did it affect the hypertrophy of myotubes as evidenced by measuring their diameters and detecting the mRNA and protein expression of hypertrophy markers. Similarly, an extracellular flux analyzer showed that Lars knockdown did not affect the metabolism (glycolysis and mitochondrial respiration) of myotubes. These results demonstrate that Lars is required for skeletal muscle differentiation through the activation of mTOR signaling, but not for hypertrophy or metabolic alteration of myotubes.

Impaired exercise tolerance, mitochondrial biogenesis, and muscle fiber maintenance in miR-133a-deficient mice.

Exercise promotes multiple beneficial effects on muscle function, including induction of mitochondrial biogenesis. miR-133a is a muscle-enriched microRNA that regulates muscle development and function. The role of miR-133a in exercise tolerance has not been fully elucidated. In the current study, mice that were deficient in miR-133a demonstrated low maximal exercise capacity and low resting metabolic rate. Transcription of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-γ coactivator 1-α, peroxisome proliferator-activated receptor-γ coactivator 1-ß, nuclear respiratory factor-1, and transcription factor A, mitochondrial were lower in miR-133a-deficient muscle, which was consistent with lower mitochondrial mass and impaired exercise capacity. Six weeks of endurance exercise training increased the transcriptional level of miR-133a and stimulated mitochondrial biogenesis in wild-type mice, but failed to improve mitochondrial function in miR-133a-deficient mice. Further mechanistic analysis showed an increase in the miR-133a potential target, IGF-1 receptor, along with hyperactivation of Akt signaling, in miR-133a-deficient mice, which was consistent with lower transcription of the mitochondrial biogenesis regulators. These findings indicate an essential role of miR-133a in skeletal muscle mitochondrial biogenesis, exercise tolerance, and response to exercise training.-Nie, Y., Sato, Y., Wang, C., Yue, F., Kuang, S., Gavin, T. P. Impaired exercise tolerance, mitochondrial biogenesis, and muscle fiber maintenance in miR-133a-deficient mice.

Incidence, Etiology, Risk Factors, and Outcomes of Bloodstream Infection after a Second Hematopoietic Stem Cell Transplantation.

Objective Infections after a second hematopoietic stem cell transplantation (HSCT) occur commonly and are associated with high mortality. However, studies on bloodstream infection (BSI) after a second HSCT are lacking. We therefore evaluated the details of BSI after a second HSCT. Methods We retrospectively evaluated the incidence, etiology, risk factors, and outcomes of BSI after a second HSCT. Patients Fifty-two adult patients with hematological malignancies who underwent allogeneic HSCT, including cord blood transplantation (CBT; n=33), as the second transplantation were enrolled. The second transplantation was limited to allogeneic HSCT. Patients who underwent HSCT for graft failure were excluded. Results The median HSCT interval was 438 (range: 39-3,893) days. Overall, 31 (59.6%) patients received autologous HSCT as the first HSCT. The cumulative incidence of BSI was 40.4% at 100 days after the second HSCT, with Gram-positive bacteria accounting for the majority (30.8%) of pathogens. Overall, 92.0% of BSIs occurred during the pre-engraftment period, and Enterococcus faecium accounted for 29.6% of pathogens. On a multivariate analysis, CBT was most closely associated with pre-engraftment BSI after the second HSCT (hazard ratio: 3.43, 95% confidence interval: 1.05-11.23, p=0.042). The 1-year survival rate after the second HSCT was lower in patients with BSI than in patients without BSI (p=0.10). Conclusion BSI is common after a second HSCT, especially with CBT. During the pre-engraftment period, BSI caused by pathogens such as E. faecium should be anticipated and appropriately treated to improve transplant outcomes.

Functional analysis reveals that Tinagl1 is required for normal muscle development in mice through the activation of ERK signaling.

Tinagl1 (tubulointerstitial nephritis antigen-like 1) is a matricellular protein involved in female infertility and breast cancer tumorigenesis. In this study, we analyzed the function of Tinagl1 in skeletal muscle using knockout mice and cell experiments. Although primary myoblasts isolated from Tinagl1-decifient (Tinagl1-/-) mice differentiated into normal myotubes, and treatment with recombinant Tinagl1 did not affect the proliferation or differentiation of C2C12 myoblasts, Tinagl1-/- mice exhibited reduced body mass and calf muscle weights compared to the control group (Tinagl1flox/flox). Furthermore, Tinagl1-/- mice showed myofibers with centrally located nuclei, which is a morphological marker of regenerating muscle or myopathy. In addition, the capillary density in the soleus muscle of Tinagl1-/- mice showed a decreasing trend compared to that of the control group. Importantly, si-RNA-mediated knockdown of TINAGL1 resulted in reduced tube formation in human umbilical vein endothelial cells (HUVECs), whereas treatment with Tinagl1 promoted tube formation. Immunoblot analysis revealed that Tinagl1 activates ERK signaling in both HUVECs and C2C12 myoblasts and myotubes, which are involved in the regulation of myogenic differentiation, proliferation, metabolism, and angiogenesis. Our results demonstrate that Tinagl1 may be required for normal muscle and capillary development through the activation of ERK signaling.

[Influence of inoculum size on MICs for methicillin-susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus].

Using 49 clinical methicillin-susceptible Staphylococcus aureus isolates (MSSA) and 54 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, we examined the change of MIC using five different inocula (2.5-4 x 10(2) cfu/spot-2.5-4 x 10(6) cfu/spot). We found the big change of the MIC with the increase of the inoculum size in ampicillin against MSSA, and the change was small in cefazolin, meropenem, ciprofloxacin. For anti-MRSA antibiotics, we found the small change with the increase of the inoculums size in vancomycin and arbekacin, and the middle change in teicoplanin and linezolid against MSSA and MRSA. The data from this study suggest that in serious and high inocula infections caused by S. aureus, the presence of an inoculum effect should be considered in curing.

Quercetin enhances fatty acid ß-oxidation by inducing lipophagy in AML12 hepatocytes.

Recent evidence demonstrated that chronic intake of quercetin attenuated hepatic fat accumulation in various animal models of obesity and diabetes. However, whether quercetin has the ability to enhance energy metabolism in hepatocytes and its exact mechanisms have yet to be identified. In the present study, we investigated whether quercetin directly enhanced the energy metabolism of cultured hepatocytes by focusing on lipophagy, involving selective autophagic degradation of lipid droplets. As an indicator of mitochondrial respiration, oxygen consumption was measured following 12-h treatment with quercetin or its related flavonoids, isorhamnetin and rutin (10 µM) using an extracellular flux analyzer. Treatment of alpha mouse liver 12 (AML12) hepatocytes with quercetin enhanced mitochondrial respiration, but isorhamnetin and rutin did not. Results of a palmitate-bovine serum albumin fatty acid oxidation assay showed that quercetin significantly increased the oxygen consumption of AML12 hepatocytes, suggesting enhanced fatty acid ß-oxidation. However, as expression levels of mitochondrial oxidative phosphorylation proteins were unaltered by quercetin, we explored whether lipophagy contributed to enhanced fatty acid ß-oxidation. Increased colocalization of lipid droplets and lysosomes confirmed that quercetin promoted lipophagy in AML12 hepatocytes. Furthermore, pharmacological inhibition of the autophagy-lysosomal pathway abolished the enhancement of fatty acid ß-oxidation induced by quercetin in AML12 hepatocytes, suggesting that the enhancement of lipophagy by quercetin contributed to increased fatty acid ß-oxidation. Finally, we showed that quercetin could activate AMPK signaling, which regulates autophagy even under nutrient-sufficient conditions. Our findings indicate that quercetin enhanced energy metabolism by a potentially novel mechanism involving promotion of lipophagy to produce the substrate for fatty acid ß-oxidation in mitochondria through activation of AMPK signaling. Our results suggest the possibility that nutrient-induced lipophagy might contributes to the reduction of fat in hepatocytes.

Energy metabolism profile of the effects of amino acid treatment on hepatocytes: Phenylalanine and phenylpyruvate inhibit glycolysis of hepatocytes.

OBJECTIVES: Amino acids are not only the building blocks of proteins, but also can be metabolized to energy substances or function as signaling molecules. The aim of this study was to profile whether amino acid treatment (essential amino acids and alanine) affects the energy metabolism (glycolysis, mitochondrial respiration) of cultured hepatocytes. METHODS: AML12 hepatocytes were treated with 5 mM of each amino acid for 1 h and the energy metabolism was then measured by using an extracellular flux analyzer. RESULTS: The results showed that phenylalanine and lysine decreased the extracellular acidification rate (ECAR), an indirect indicator of glycolysis, whereas isoleucine and histidine increased the ECAR. Amino acids did not affect the oxygen consumption rate, an indirect indicator of mitochondrial respiration. The glycolysis stress test revealed that treatment of the hepatocytes with phenylalanine inhibited glycolysis when the concentration of the substrate for glycolysis was sufficient in cultured media. We also investigated the effect of metabolites derived from conversion of phenylalanine on glycolysis in hepatocytes and found that phenylpyruvate inhibited glycolysis, whereas tyrosine and phenylethylamine did not affect glycolysis. CONCLUSIONS: The findings from the present study complement basic knowledge of the effects of amino acid treatment on energy metabolism in cultured hepatocytes and indicate that phenylalanine and phenylpyruvate inhibit glycolysis.

Listeria monocytogenes Bacteremia During Isatuximab Therapy in a Patient with Multiple Myeloma.

An elderly patient with multiple myeloma (MM) was being treated with several regimens and developed a severe drug eruption, necessitating the use of atovaquone instead of trimethoprim-sulfamethoxazole for pneumocystis pneumonia (PCP) prophylaxis. For progressive MM, treatment with isatuximab, an anti-CD38 monoclonal antibody, was started. During the treatment, he developed Listeria monocytogenes bacteremia and recovered quickly with ampicillin administration. CD38 is closely related to the innate immune response against L. monocytogenes, and isatuximab may increase the risk of infection. Therefore, trimethoprim-sulfamethoxazole may be useful in the prevention of not only PCP but also L. monocytogenes infection.

Data on the proliferation and differentiation of C2C12 myoblast treated with branched-chain ketoacid dehydrogenase kinase inhibitor.

The catabolism of branched chain amino acids (BCAAs) is mainly carried out in skeletal muscle myofibers. It is mediated by branched chain aminotransferase 2 and branched chain alpha ketoacid dehydrogenase (BCKDH) in mitochondria for energy supply, especially during exercise. BCKDH kinase (BCKDK) is a negative regulator of BCAAs catabolism by its inhibitory phosphorylation of the BCKDH E1a subunit. The data presented in this article are related to the research article that we previously have reported entitled "Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes" (Suzuki et al., 2020)[1]. In this report, we have demonstrated that 1hour treatment of BT2, an inhibitor of BCKDK, decreased the glycolysis of C2C12 differentiated myotubes compared to the control. Although BCAAs metabolism is basically assumed to be carried out in differentiated myofibers, BCKDK is expressed in both undifferentiated myoblasts and differentiated myotubes, and the biological and physiological significance of BCAAs metabolism in myoblasts is still unclear. Present data demonstrate an in vitro assessment of BT2 on C2C12 myoblasts proliferation and differentiation. The data suggest that activation of BCAAs catabolism by the BCKDK inhibitor BT2 impairs C2C12 myoblasts proliferation and differentiation.

Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes.

OBJECTIVES: Amino acids are not only components of proteins, but also can be metabolized to energy substances or be used as signaling molecules. However, basic knowledge of the relationship between amino acid treatment and energy metabolism is still insufficient. The aims of this study was to profile the effects of essential amino acid and alanine treatment on the energy metabolism of both myoblasts and myotubes and to contribute to the understanding of the basic relationship between amino acid treatment and energy metabolism of skeletal muscle cell. METHODS: We profiled whether amino acid (essential amino acids and alanine) treatment can affect the energy metabolism (glycolysis, mitochondrial respiration) of cultured skeletal muscle cells. C2C12 myoblasts and differentiated myotubes were treated with 5 mM each amino acid for 1 h, then the energy metabolism was measured by using extracellular flux analyzer. RESULTS: Although not all of the amino acid treatments could affect the energy metabolism of C2C12 myoblasts, leucine, isoleucine, lysine, phenylalanine, and histidine decreased the extracellular acidification rate, an indirect indicator of glycolysis, in differentiated myotubes without alteration of oxygen consumption rate, an indirect indicator of mitochondrial respiration. By glycolysis stress test, we found that leucine treatment inhibited glycolysis of myotubes when the substrate of glycolysis is sufficient in cultured media. The inhibitory effect of glycolysis by leucine was not canceled by rapamycin (an inhibitor for mTOR). But, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (an inhibitor for branched-chain α ketoacid dehydrogenase complex kinase) increased branched-chain amino acid catabolism, which decreased the glycolysis of myotubes. CONCLUSION: Findings from the present study complemented the basic knowledge of amino acid treatment on the energy metabolism of cultured skeletal muscle cells and suggested the inhibitory effects of glycolysis by branched-chain amino acid catabolism.

Acute oral administration of L-leucine upregulates slow-fiber- and mitochondria-related genes in skeletal muscle of rats.

Branched-chain amino acids promote both protein and mRNA synthesis through mechanistic target of rapamycin (mTOR) signaling. A previous report demonstrated that chronic branched-chain amino acid supplementation increased mitochondrial biogenesis in the skeletal muscle of middle-aged mice through activation of mTOR signaling. In this study, we hypothesized that the acute oral administration of L-leucine alone has the ability to alter the gene expression related to fiber type and metabolism in skeletal muscle of young rats through the activation of mTOR signaling. Although the gene expression of representative glycolytic enzymes (Hk2 and Eno3) was not altered, L-leucine administration (135 mg/100 g body weight) upregulated the expression of slow-fiber-related genes (Myh7, Myl3, and Tnni1) and a mitochondrial biogenesis-related gene (Ppargc1a) in the soleus and extensor digitorum longus muscles compared with the control. In addition, L-leucine treatment also upregulated the slow-fiber genes and mitochondrial gene expression in cultured C2C12 myotubes, whereas rapamycin inhibited the effects of L-leucine. However, L-alanine, L-phenylalanine, and L-valine treatment did not alter the expression of the fiber type- and metabolism-related genes as observed in L-leucine. Our results suggest that L-leucine may have the ability to alter skeletal muscle fiber type toward slow fiber and oxidative metabolism by upregulation of gene expression through mTOR signaling.

Glucosamine abrogates the stem cell factor + endothelin-1-induced stimulation of melanogenesis via a deficiency in MITF expression due to the proteolytic degradation of CREB in human melanocytes.

We have already reported that glucosamine (GlcN) distinctly abrogates the pigmentation of human epidermal equivalents stimulated by stem cell factor + endothelin-1 (SE). In this study, we characterized the molecular mechanism involved in the anti-melanogenic effects of GlcN using normal human melanocytes (NHMs) in culture. The SE-stimulated gene (12 h) and protein (24 h) expression levels of melanocyte-specific proteins (at the indicated times post-stimulation) were significantly abrogated by pretreatment with GlcN for 72 h. Western blotting analysis of the phosphorylation of intracellular signaling molecules in the MAPK pathway revealed that despite the significantly decreased level of total CREB protein at all times post-stimulation, the SE-stimulated phosphorylation of ERK, CREB and MITF is not attenuated at 15 min post-stimulation in GlcN-treated NHMs. However, the SE-stimulated protein expression level of total MITF at 2 and 6 h post-stimulation was significantly abrogated by 72 h pretreatment with GlcN. Consistently, pretreatment with GlcN for 72 h abrogated the stimulated gene and protein expression levels of MITF at 1 h and 2 h post-stimulation, respectively. Analysis of gene and protein expression levels also demonstrated that pretreatment with GlcN for 72 h significantly reduced the protein levels of CREB and MITF without affecting their gene expression levels prior to the SE stimulation. Silencing with a CREB siRNA distinctly abrogated the SE-stimulated expression of MITF (at 2 h post-stimulation) and melanocyte-specific proteins (at 24 h post-stimulation). Similarly, transfection of MITF siRNA markedly abrogated the SE-stimulated expression of MITF protein and melanocyte-specific proteins at 2 and 24 h post-stimulation, respectively. Finally, the decreased levels of CREB and MITF proteins induced by 72 h pretreatment with GlcN were abrogated by the co-addition of the proteosomal degradation inhibitor MG132. These findings suggest that the anti-melanogenic effect elicited by GlcN is mediated via the decreased expression of MITF which results from the attenuated transcriptional activity of CREB due to proteolytic degradation.