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1.  Pyrithione-zinc Prevents UVB-induced Epidermal Hyperplasia by Inducing HIF-1α 
Epidermal keratinocytes overgrow in response to ultraviolet-B (UVB), which may be associated with skin photoaging and cancer development. Recently, we found that HIF-1α controls the keratinocyte cell cycle and thereby contributes to epidermal homeostasis. A further study demonstrated that HIF-1α is down-regulated by UVB and that this process is involved in UVB-induced skin hyperplasia. Therefore, we hypothesized that the forced expression of HIF-1α in keratinocytes would prevent UVB-induced keratinocyte overgrowth. Among several agents known to induce HIF-1α, pyrithione-zinc (Py-Zn) overcame the UVB suppression of HIF-1α in cultured keratinocytes. Mechanistically, Py-Zn blocked the degradation of HIF-1α protein in keratinocytes, while it did not affect the synthesis of HIF-1α. Moreover, the p21 cell cycle inhibitor was down-regulated after UVB exposure, but was robustly induced by Py-Zn. In mice repeatedly irradiated with UVB, the epidermis became hyperplastic and HIF-1α disappeared from nuclei of epidermal keratinocytes. However, a cream containing Py-Zn effectively prevented the skin thickening and up-regulated HIF-1α to the normal level. These results suggest that Py-Zn is a potential agent to prevent UVB-induced photoaging and skin cancer development. This work also provides insight into a molecular target for treatment of UVB-induced skin diseases.
PMCID: PMC2869458  PMID: 20473380
Ultraviolet; Skin; Hyperplasia; Hypoxia-inducible factor-1α; Pyrithione-zinc
2.  STAT3 inhibits the degradation of HIF-1α by pVHL-mediated ubiquitination 
Experimental & Molecular Medicine  2008;40(5):479-485.
Hypoxia-inducible factor 1α (HIF-1α) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1α is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1α protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1α stability. We found that STAT3 interacts with C-terminal domain of HIF-1α and stabilizes HIF-1α by inhibition of pVHL binding to HIF-1α. The binding between HIF-1α and pVHL, negative regulator of HIF-1α stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1α protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1α protein via inhibition of interaction between pVHL and HIF-1α. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1α through competition with pVHL for binding to HIF-1α, and then stabilizes HIF-1α protein levels.
PMCID: PMC2679355  PMID: 18985005
anoxia; hypoxia-inducible factor1, α subunit; neoplasms; STAT3 transcription factor; ubiquitination; von Hippel-Lindau tumor suppressor protein
3.  Antihyperglycemic mechanism of metformin occurs via the AMPK/LXRα/POMC pathway 
Scientific Reports  2015;5:8145.
Metformin is a first-line drug for treating type 2 diabetes. Although metformin is known to phosphorylate AMP-activated protein kinase (AMPK), it is unclear how the glucose-lowering effect of metformin is related to AMPK activation. The aim of this study was to identify the urinary endogenous metabolites affected by metformin and to identify the novel underlying molecular mechanisms related to its anti-diabetic effect. Fourteen healthy male subjects were orally administered metformin (1000 mg) once. First morning urine samples were taken before and after administration to obtain metabolomic data. We then further investigated the anti-diabetic mechanism of metformin in vitro and in vivo. The fluctuation of the metabolite cortisol indicated that the neuroendocrine system was involved in the anti-diabetic effect of metformin. Actually we found that metformin induced AMPK/liver X receptor α (LXRα) phosphorylation, followed by pro-opiomelanocortin (POMC) suppression in rat pituitary cells. We confirmed this result by administering metformin in an animal study. Given that cortisol stimulates gluconeogenesis, we propose the anti-hyperglycemic effect of metformin is attributed to reduced POMC/adrenocorticotropic hormone (ACTH)/cortisol levels following AMPK/LXRα phosphorylation in the pituitaries.
PMCID: PMC4311245  PMID: 25634597
4.  Long-Term Outcome of Low-Energy Extracorporeal Shock Wave Therapy for Plantar Fasciitis: Comparative Analysis According to Ultrasonographic Findings 
Annals of Rehabilitation Medicine  2014;38(4):534-540.
To investigate the long-term effect of low-energy extracorporeal shock wave therapy (ESWT) for plantar fasciitis (PF) according to ultrasonography (US) findings.
Thirty feet of 25 patients with clinical diagnosis of PF were enrolled and divided into two groups (Apparent-US and Uncertain-US) according to US findings, such as plantar fascia thickening or hypoechogenicity. Inclusion criteria were symptom duration >6 months and a fair or poor grade in Roles-Maudsley score (RMS). ESWT (0.10 mJ/mm2, 600 shocks) was given once a week for 6 weeks. Numeric rating scale (NRS) and RMS were evaluated prior to each ESWT session, at short-term follow-up (one week after all ESWT sessions) and long-term follow-up telephone interview (mean 24 months after ESWT). Good and excellent grade in RMS were considered as treatment success.
Repeated measure ANOVA demonstrated that NRS significantly decreased with time after ESWT up to the long-term follow-up (time effect, p<0.001) without group-time interaction (p=0.641), indicating that ESWT equally decreased pain in both groups. Overall success rate was 63.3% (short-term follow-up) and 80.0% (long-term follow-up). In comparative analysis between groups, success rate of Apparent-US and Uncertain-US at short-term follow-up was 61.9% and 66.7%, respectively, and 85.7% and 66.7%, respectively, at long-term follow-up.
If other causes of heel pain are ruled out through meticulous physical examination and ultrasonography, low-energy ESWT in PF seems to be beneficial regardless of US findings. In terms of success rate, however, long-term outcome of Apparent-US appears to be superior to Uncertain-US.
PMCID: PMC4163593  PMID: 25229032
Plantar fasciitis; Extracorporeal shock wave therapy (ESWT); Ultrasonography; Treatment outcome
5.  Angiopoietin-1 Elicits Pro-Inflammatory Responses in Monocytes and Differentiating Macrophages 
Molecules and Cells  2013;35(6):550-556.
The angiopoietin/Tie2 system is an important regulator of angiogenesis and inflammation. In addition to its functions in endothelial cells, Tie2 expression on non-endothelial cells allows for angiopoietin ligands to stimulate the cells. Although Ang1 is a strong Tie2 receptor agonist, little is known regarding the effect of Ang1 on non-endothelial cells, such as monocytes and macrophages. In this study, we found that Ang1 functionally binds to and stimulates monocytes via p38 and Erk1/2 phosphorylation. Ang1-mediated monocyte stimulation is associated with pro-inflammatory cytokine TNF-α expression. We also determined that Ang1 switched macrophage differentiation toward a pro-inflammatory phenotype, even in the presence of an anti-inflammatory mediator. These findings suggest that Ang1 plays a role in stimulating pro-inflammatory responses and could provide a new strategy by which to manage inflammatory responses.
PMCID: PMC3887877  PMID: 23686433
angiopoietin; inflammation; macrophage; monocyte; Tie2
6.  Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF 
EMBO Molecular Medicine  2012;4(9):924-938.
Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed ‘hypoxic-priming’) efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation.
PMCID: PMC3491825  PMID: 22821840
embryoid bodies; endothelial cells; mesoderm differentiation; mouse embryonic stem cells; niche
7.  Heterodimerization of Glycosylated Insulin-Like Growth Factor-1 Receptors and Insulin Receptors in Cancer Cells Sensitive to Anti-IGF1R Antibody 
PLoS ONE  2012;7(3):e33322.
Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.
Methodology/Principal Findings
In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.
Conclusion and Significance
The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.
PMCID: PMC3306383  PMID: 22438913
8.  Constitutive phosphorylation of the FOXO1 transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis-related molecules 
BMC Cancer  2011;11:264.
Although FOXO transcription factors may have an anti-angiogenic role, little is known about their role in tumor angiogenesis. The present study was performed to investigate the correlation between the constitutive expression of phosphorylated FOXO1 (pFOXO1) and angiogenesis in gastric cancer.
Immunohistochemistry was performed on tissue array slides containing 272 gastric carcinoma specimens, and the correlations between the cytoplasmic pFOXO1 expression in gastric cancer cells and CD34-immunopositive microvessel area (MVA) or the expressions of angiogenesis-related molecules were analyzed. In vitro analyses with Western blotting and semiquantitative reverse transcription-polymerase chain reaction were performed using the stable SNU-638 gastric cancer cell line transfected with lentivirus-delivered FOXO1 short hairpin RNA.
The cytoplasmic expression of pFOXO1 in tumor cells was observed in 85% of gastric carcinoma cases, and was found to be positively associated with higher MVA (P = 0.048). Moreover, pFOXO1 expression was positively correlated with the expressions of several angiogenesis-related proteins, including hypoxia inducible factor-1α (HIF-1α, P = 0.003), vessel endothelial growth factor (P = 0.004), phosphorylated protein kinase B (P < 0.001), and nuclear factor-κB (P = 0.040). In contrast, the expression of pFOXO1 was not correlated with that of phosphorylated signal transducer and activator of transcription 3 or β-catenin. In addition, cell culture experiments showed that FOXO1 suppression increased the mRNA and protein expressions of HIF-1α.
Our results suggest that pFOXO1 expression in cancer cells plays a role in gastric cancer angiogenesis via mechanisms involving various angiogenesis-related molecules. Animal experiments are needed to confirm the anti-angiogenic role of FOXO1 in human gastric cancer.
PMCID: PMC3135570  PMID: 21696576
pFOXO1; angiogenesis; gastric cancer; immunohistochemistry; tissue array analysis
The Journal of experimental biology  1998;201(Pt 8):1197-1201.
The physiological regulation of the red cell mass depends upon enhanced transcription of the erythropoietin (Epo) gene in response to hypoxia. Studies of Epo gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia and respond with biologically appropriate alterations in gene expression. It is likely that oxygen sensing involves a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. Indirect evidence suggests that the sensor is present in all cells and is a multi-subunit assembly containing an NAD(P)H oxidase capable of generating peroxide and reactive oxygen intermediates, which serve as signaling molecules. The up-regulation of Epo gene transcription by hypoxia is mediated by at least two known DNA-binding transcription factors, hypoxia-inducible factor 1 (HIF-1) and hepatic nuclear factor 4 (HNF-4), which bind to cognate response elements in a critical 3′ enhancer approximately 50 bp in length. HIF-1 binding is induced by hypoxia as well as by cobalt. The activation of HIF-1 by hypoxia depends upon the selective protection of its α subunit from ubiquitin-dependent proteolysis by means of a mechanism that involves redox chemistry and perhaps phosphorylation. HNF-4 is an orphan nuclear receptor that is constitutively expressed in kidney and liver and which cooperates with HIF-1 to give maximal hypoxic induction. In hypoxic cells, p300 or a related family member forms a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3′ enhancer to the apparatus on the promoter responsible for the initiation of transcription.
PMCID: PMC3044471  PMID: 9510530
erythropoietin; hypoxia; gene regulation; oxygen sensing; HIF-1; HNF-4; p300
10.  Constitutive activation of glycogen synthase kinase-3β correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer 
BMC Gastroenterology  2010;10:91.
Aberrant regulation of glycogen synthase kinase-3β (GSK-3β) has been implicated in several human cancers; however, it has not been reported in the gastric cancer tissues to date. The present study was performed to determine the expression status of active form of GSK-3β phosphorylated at Tyr216 (pGSK-3β) and its relationship with other tumor-associated proteins in human gastric cancers.
Immunohistochemistry was performed on tissue array slides containing 281 human gastric carcinoma specimens. In addition, gastric cancer cells were cultured and treated with a GSK-3β inhibitor lithium chloride (LiCl) for immunoblot analysis.
We found that pGSK-3β was expressed in 129 (46%) of 281 cases examined, and was higher in the early-stages of pathologic tumor-node-metastasis (P < 0.001). The expression of pGSK-3β inversely correlated with lymphatic invasion (P < 0.001) and lymph node metastasis (P < 0.001) and correlated with a longer patient survival (P < 0.001). In addition, pGSK-3β expression positively correlated with that of p16, p21, p27, p53, APC, PTEN, MGMT, SMAD4, or KAI1 (P < 0.05), but not with that of cyclin D1. This was confirmed by immunoblot analysis using SNU-668 gastric cancer cells treated with LiCl.
GSK-3β activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis. Thus, these findings suggest that GSK-3β activation is a useful prognostic marker for the early-stage gastric cancer.
PMCID: PMC2928182  PMID: 20704706
11.  Arrest Defective-1 Controls Tumor Cell Behavior by Acetylating Myosin Light Chain Kinase 
PLoS ONE  2009;4(10):e7451.
The enhancement of cell motility is a critical event during tumor cell spreading. Since myosin light chain kinase (MLCK) regulates cell behavior, it is regarded as a promising target in terms of preventing tumor invasion and metastasis. Since MLCK was identified to be associated with human arrest defective-1 (hARD1) through yeast two-hybrid screening, we here tested the possibility that hARD1 acts as a regulator of MLCK and by so doing controls tumor cell motility.
Methodology/Principal Findings
The physical interaction between MLCK and hARD1 was confirmed both in vivo and in vitro by immunoprecipitation assay and affinity chromatography. hARD1, which is known to have the activity of protein lysine ε-acetylation, bound to and acetylated MLCK activated by Ca2+ signaling, and by so doing deactivated MLCK, which led to a reduction in the phosphorylation of MLC. Furthermore, hARD1 inhibited tumor cell migration and invasion MLCK-dependently. Our mutation study revealed that hARD1 associated with an IgG motif of MLCK and acetylated the Lys608 residue in this motif. The K608A-mutated MLCK was neither acetylated nor inactivated by hARD1, and its stimulatory effect on cell motility was not inhibited by hARD1.
These results indicate that hARD1 is a bona fide regulator of MLCK, and that hARD1 plays a crucial role in the balance between tumor cell migration and stasis. Thus, hARD1 could be a therapeutic target in the context of preventing tumor invasion and metastasis.
PMCID: PMC2758594  PMID: 19826488
12.  Non-hypoxic transcriptional activation of the aryl hydrocarbon receptor nuclear translocator in concert with a novel hypoxia-inducible factor-1alpha isoform 
Nucleic Acids Research  2004;32(18):5499-5511.
Aryl hydrocarbon receptor nuclear translocator (ARNT) belongs to the basic helix–loop–helix Per-Arnt-Sim (bHLH PAS) protein which dimerizes with other PAS proteins. Although it has a transactivation domain (TAD), ARNT functions as an assistant partner of main factors, such as aryl hydrocarbon receptor and hypoxia-inducible factors, rather than acting as a straightforward transcription factor. However, ARNT may function as an active transcription factor using its TAD either in association with itself, single-minded protein 1, or trachealess protein. In the present study, we identified a novel ARNT partner, a HIF-1α variant, which is ubiquitously expressed in human tissues and cancer cell lines. The HIF-1α variant, designated HIF-1α417, bound to ARNT and, moreover, stimulated the transcription of the erythropoietin enhancer reporter gene. This stimulation was markedly augmented by ARNT but not by the ARNT603 mutant lacking the TAD. Thus, augmentation by ARNT suggests that ARNT determined the transcriptional activity. HIF-1α417 was found to be associated with ARNT and to bind to the hypoxia response element containing the E-box core. Moreover, HIF-1α417 promoted the nuclear translocation of ARNT, and conversely ARNT stabilized HIF-1α417. Taken together, our results suggest that HIF-1α417 is a novel partner that is required for transcription activity of ARNT.
PMCID: PMC524291  PMID: 15479785
13.  Oxygen-dependent and -independent regulation of HIF-1alpha. 
Journal of Korean Medical Science  2002;17(5):581-588.
Hypoxia-inducible factor-1 (HIF-1) is composed of HIF-1alpha and HIF-1beta, and is a master regulator of oxygen homeostasis, playing critical roles in physiological and pathological processes. Normally, the formation and transcriptional activity of HIF-1 depend on the amount of HIF-1alpha, and the expression of HIF-1alpha is tightly controlled by the cellular oxygen tension. Recent progress in the study of its regulation mechanism provided clues as to how HIF-1alpha is regulated by oxygen. It appears that HIF-1alpha is not regulated only by the oxygen tension, but also by various other stimuli, such as transition metals, nitric oxide, reactive oxygen species, growth factors, and mechanical stresses. In this review, we summarize the oxygen-dependent and -independent regulation of HIF-1alpha, and the respective physiological and pathological meanings.
PMCID: PMC3054927  PMID: 12378005

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