Effect of low skeletal muscle mass and sarcopenic obesity on chronic kidney disease in patients with type 2 diabetes.

OBJECTIVE: This study aimed to investigate the association between low muscle mass or sarcopenic obesity and the risk of incident chronic kidney disease (CKD) in patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 3123 patients with T2DM with preserved renal function were followed up for incident CKD. Skeletal muscle mass was estimated from bioelectrical impedance analysis. CKD was defined as estimated glomerular filtration rate < 60 mL/min/1.73 m2 . Sarcopenic obesity was defined as the coexistence of sarcopenia and abdominal obesity. RESULTS: During 8.9 years of follow-up, 530 (17.0%) patients developed incident CKD. When patients were divided into three groups based on sex-specific tertiles, lower muscle mass was not associated with an increased risk of incident CKD after adjustment for risk factors. However, when patients were divided into four groups according to the presence of sarcopenia and obesity, sarcopenic obesity was associated with an increased risk of incident CKD (adjusted hazard ratio 1.77; 95% CI: 1.24-2.51; p = 0.001) compared with the other groups. CONCLUSIONS: Sarcopenic obesity, but not low muscle mass alone, may increase the risk of CKD in patients with T2DM.

Effect of Dapagliflozin in Combination with Lobeglitazone and Metformin in Korean Patients with Type 2 Diabetes in Real-World Clinical Practice.

PURPOSE: This study aimed to evaluate the efficacy and tolerability of dapagliflozin as an add-on or a switch therapy to lobeglitazone plus metformin (MFM) in Korean patients with inadequately controlled type 2 diabetes mellitus (T2DM) in real-world clinical practice. MATERIALS AND METHODS: The study included 109 patients who started dapagliflozin as add-on or switch therapy to lobeglitazone plus MFM. The primary outcome was a change in glycated hemoglobin (HbA1c) level from baseline after 12 months of treatment. Secondary outcomes included changes in fasting plasma glucose (FPG), lipid profiles, body weight, visceral fat area (VFA), and blood pressure after 12 months of treatment. RESULTS: The baseline HbA1c was 8.3±1.3% (8.7±1.5% in the add-on group and 8.1±1.0% in the switch group). After 12 months, mean HbA1c decreased (-0.91%) in all patients (p<0.05) (-1.39% in the add-on group and -0.63% in the switch group). Significant reductions in FPG were also observed in both the add-on and switch groups (-54.37 mg/dL and -24.68 mg/dL, respectively). Overall, there was a significant improvement in serum triglyceride (-24.74 mg/dL), low density lipoprotein cholesterol (-7.92 mg/dL), body weight (-2.98 kg), VFA (-9.00 cm²), and systolic blood pressure (-8.67 mm Hg). Approximately 35.8% of patients achieved HbA1c <7.0% after 12 months. CONCLUSION: Dapagliflozin, as an add-on or a switch therapy to lobeglitazone plus MFM, can be a suitable alternative for Korean patients with inadequately controlled T2DM. The combination therapy resulted in significant reductions in HbA1c levels, body weight, and blood pressure.

Advanced Liver Fibrosis Is Associated with Chronic Kidney Disease in Patients with Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). However, the causal relationship between NAFLD and CKD is uncertain, particularly in patients with type 2 diabetes mellitus (T2DM). We aimed to investigate the association between the presence and severity of NAFLD and incident CKD in patients with T2DM. METHODS: In this longitudinal cohort study of patients with T2DM, 3,188 patients with preserved renal function were followed up for the occurrence of incident CKD. NAFLD was defined as the presence of hepatic steatosis on ultrasonography, without any other causes of chronic liver disease. Advanced liver fibrosis of NAFLD was defined as a fibrosis-4 index ≥2.67. CKD was defined as an estimated glomerular filtration rate <60 mL/min/1.73 m2. RESULTS: At baseline, 1,729 (54.2%) patients had NAFLD, of whom 94 (5.4%) had advanced liver fibrosis. During the follow-up of 8.3±3.6 years, 472 (14.8%) patients developed incident CKD: 220 (15.1%) in the non-NAFLD group, 231 (14.1%) in the NAFLD without advanced fibrosis group and 28 (31.1%) in the NAFLD with advanced fibrosis group. There was no increased risk of incident CKD in the NAFLD group compared to the non-NAFLD group (P=0.435). However, among patients with NAFLD, advanced liver fibrosis was associated with an increased risk of CKD (adjusted hazard ratio, 1.75; 95% confidence interval, 1.15 to 2.66; P=0.009). CONCLUSION: Advanced liver fibrosis in patients with NAFLD is independently associated with an increased risk of incident CKD in patients with T2DM.

Functional impairment of CD19+CD24hiCD38hi B cells in neuromyelitis optica spectrum disorder is restored by B cell depletion therapy.

The role of B cells in immune response regulation is context dependent. In some cases, bystander B cell activation leads to interleukin-10 (IL-10) production, suppressing inappropriate immune responses. However, the role of B cells in regulation of autoimmune diseases, including neuromyelitis optica spectrum disorder (NMOSD), is incompletely understood. NMOSD is an autoimmune disease of the central nervous system with a relapsing-remitting course in which acute attacks lead to severe disability. B cell depletion therapy (BCDT) has shown clinical efficacy in NMOSD by eliminating pathogenic B cells; however, its effect on regulatory B (Breg) cells remains elusive. Here, we evaluated the B cell subsets, Breg cell function, and the effect of BCDT on these cells in patients with NMOSD. We showed that CD24hiCD38hi B cells from patients with NMOSD did not inhibit CD4+ T cell production of interferon-γ (IFN-γ), IL-17, or IL-21 and failed to inhibit follicular helper T cell expansion or induce regulatory T cells. This cellular impairment in patients with NMOSD can be explained by deficient Breg cell numbers and Breg cell­intrinsic deficits in IL-10 production specifically in response to B cell bystander activation. Using cross-sectional and 3-year longitudinal studies, we showed that BCDT treatment restored the numerical deficiency of Breg cells. Moreover, the post-BCDT repopulated CD24hiCD38hi B cells restored IL-10 production and suppressed IFN-γ and IL-17 production by CD4+ T cells. Our results suggest that both numerical deficiency of CD24hiCD38hi B cells and their impaired regulatory function contribute to NMOSD pathophysiology, and function is restored after BCDT.

Efficacy and Cerebrospinal Fluid Rhinorrhea after Cabergoline Treatment in Patients with Bioactive Macroprolactinoma.

Predicting dopamine agonist resistance in patients with macroprolactinoma is essential for clinicians to prevent treatment failure and subsequent complications such as medication-induced cerebrospinal fluid (CSF) rhinorrhea. We evaluated the features of patients with cabergoline resistance and CSF rhinorrhea in patients with prolactinomas with prolactin levels ≥1000 ng/mL. A total of 140 patients who were newly diagnosed with prolactinoma secreting only prolactin ≥1000 ng/mL and treated with cabergoline for the first time were included in this study. Based on the hormonal and radiologic response of the prolactinoma, the patients were divided into responders and non-responders. Non-responders (36/140, 25.8%) included a higher number of patients receiving hormone replacement than responders (responders, n (%) = 12(11.5) vs. non-responders = 13(36.1), p = 0.001). In propensity score matching analysis, patients who developed CSF rhinorrhea presented more frequent hormone deficiency than responders regardless of initial cabergoline dose. Hormone deficiency was associated with a greater odds ratio for the risk of non-responders (adjusted odds ratio = 5.13, 95% CI 1.96-13.46, p = 0.001). Cabergoline was effective in bioactive macroprolactinoma. Furthermore, initial cabergoline dose was not significantly associated with long-term responsiveness and development of CSF rhinorrhea but the hypopituitarism was independently associated with an increased risk of cabergoline resistance and CSF rhinorrhea.

Low muscle mass is associated with carotid atherosclerosis in patients with type 2 diabetes.

BACKGROUND AND AIMS: Sarcopenia leads to metabolic and vascular abnormalities. However, little is known regarding the independent relationship between skeletal muscle mass and atherosclerosis in patients with type 2 diabetes mellitus (T2DM). This study aimed to evaluate the association between skeletal muscle mass and carotid atherosclerosis in men and women with T2DM. METHODS: In this cross-sectional study, a total of 8202 patients with T2DM were recruited from the Seoul Metabolic Syndrome cohort. Skeletal muscle mass was estimated using bioimpedance analysis, while skeletal muscle mass index (SMI, %) was defined as total skeletal muscle mass (kg)/body weight (kg) × 100. Both carotid arteries were examined by B-mode ultrasound. Carotid atherosclerosis was defined by having a carotid plaque or mean carotid intima-media thickness (IMT) ≥1.1 mm. RESULTS: Among the entire population, 4299 (52.4%) subjects had carotid atherosclerosis. The prevalence of carotid atherosclerosis increased with decreasing SMI quartiles for both sexes. The odds ratios for carotid atherosclerosis were 2.33 (95% confidence interval [CI], 1.17-4.63) and 2.24 (95% CI, 1.06-4.741) in the lowest versus highest SMI quartile in men and women, respectively, after the adjustment for clinical risk factors. In men, the risk of atherosclerosis increased linearly with decreasing SMI quartiles (p for trend = 0.036). CONCLUSIONS: Low skeletal muscle mass was independently associated with the presence of carotid atherosclerosis in men and women with T2DM.

Restoration of regulatory B cell deficiency following alemtuzumab therapy in patients with relapsing multiple sclerosis.

BACKGROUND: Regulatory B cells (Bregs), which protect from autoimmunity, are deficient in multiple sclerosis (MS). Novel regulatory B cell subsets CD19+CD24hiCD38hi cells and CD19+PD-L1hi cells, with disparate regulatory mechanisms have been defined. Alemtuzumab provides a long-lasting suppression of disease activity in MS. In contrast to its documented efficacy, alemtuzumab's mechanism of action is not fully understood and information about the composition of repopulating B cell pool is scarce. AIM: To characterize repopulated B cell subsets and elucidate alemtuzumab's mechanism of action in B cell perspective. METHODS: The frequency and the absolute number of Bregs were studied in peripheral blood mononuclear cells (PBMC) of 37 MS patients and 11 healthy controls (HC). Longitudinal analysis of the frequency and the absolute number of Bregs in PBMC of 11 MS patients was evaluated, before and at 6, 9, and 12 months post alemtuzumab. RESULTS: We found deficiency of CD19+CD24hiCD38hi cells during relapse compared to remission and HC (relapse vs remission: p = 0.0006, relapse vs HC: p = 0.0004). CD19+PD-L1hi cells were deficient during relapse than remission and HC (relapse vs remission: p = 0.0113, relapse vs HC: p = 0.0007). Following alemtuzumab, the distribution of B cells shifts towards naïve phenotype and Breg deficiency is restored. The frequency of CD19+CD24hiCD38hi cells was significantly increased at 6 M and 9 M compared to 0 M (6 M vs 0 M: p = 0.0004, 9 M vs 0 M: p = 0.0079). At 9 M, the frequency of CD19+CD24hiCD38hi cells started to decrease and by 12 M the frequency was reduced compared to 6 M, although it was significantly higher than baseline level (12 M vs 0 M: p = 0.0257). The absolute number was significantly increased at 6 M and 9 M post-alemtuzumab (6 M vs 0 M: p = 0.0063, 9 M vs 0 M: p = 0.02). The frequency of CD19+PD-L1hi cells significantly increased until 12 M (6 M vs 0 M: p = 0.0004, 12 M vs 0 M: p = 0.0036). The frequency of CD19+PD-L1hi cells at 12 M was significantly higher than 9 M (p = 0.0311). We further pinpoint that CD19+CD24hiCD38hi cells were deficient at severe relapses following alemtuzumab infusion and restored during recovery. CONCLUSIONS: Our results highlight the preferential reconstitution of Bregs as a possible mechanism of action of alemtuzumab and CD19+CD24hiCD38hi cells as a potential biomarker for disease activity.

Foxl2, a forkhead transcription factor, modulates nonclassical activity of the estrogen receptor-alpha.

Foxl2 is a forkhead transcription factor required for ovary development and ovarian follicle maturation. In this report, we identified and characterized a functional relationship between Foxl2 expression and estrogen receptor (ER)-alpha signaling. We show that Foxl2 has no effect on classical ERalpha-mediated transcription, which occurs through canonical estrogen response elements. However, Foxl2 suppresses ERalpha signaling through nonclassical tethered transcriptional pathways. Specifically, the selective ER modulator tamoxifen stimulates activator protein-1 (AP1)-dependent transcription via the ERalpha, and this enhancement is blocked by Foxl2. Two lines of evidence suggest that Foxl2 suppression is mediated by physical interactions with ERalpha rather than direct action at AP1 binding sites. First, ERalpha is coimmunoprecipitated with Foxl2. Second, activation of a upstream activating sequence (UAS) reporter by Gal4-cJun in the presence of ERalpha and tamoxifen was blocked by Foxl2, demonstrating suppression in the absence of an AP1 site. Cyclooxygenase-2 (COX2), which is required for ovulation, was identified through expression profiling as a candidate physiological target for nonclassical ERalpha signaling and thus modulation by ERalpha/Foxl2 interactions. This possibility was confirmed by two sets of experiments. COX2 protein levels were induced by ERalpha in the presence of tamoxifen, and protein expression was suppressed by Foxl2. In addition, ERalpha stimulation of the COX2 promoter was repressed by Foxl2. We conclude that ERalpha and Foxl2 interact and that Foxl2 selectively suppresses ERalpha-mediated transcription of AP1-regulated genes. These data provide a potential point of convergence for ERalpha and Foxl2 to regulate ovarian development and function.

A phenotypic spectrum of sexual development in Dax1 (Nr0b1)-deficient mice: consequence of the C57BL/6J strain on sex determination.

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.

TGFbeta plasmid construction and delivery for the prevention of type 1 diabetes.

Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet beta cell destruction can be manipulated by the administration of Th(2) cytokines. Using gene delivery to express the targeted protein, we can overcome the need for frequent administration of cytokines on account of their short half-lives. In this study, the effect of hTGFbeta gene delivery was evaluated both in vitro and in vivo using an adenovirus vector (Ad) constructed with an hTGFbeta cDNA. In vitro transfection assays of the construct in HepG2, beta cell lines, and islets showed good expression levels of hTGFbeta and activation of smad3. Ad-hTGFbeta enhanced differentiation and proliferation in the beta cell line or islets without causing apoptosis. Of interest, Ad-hTGFbeta transduction in CD4(+)CD25(-) T cells resulted in a significant enhanced expression of CD25 and a regulatory T cell-specific transcription factor, Foxp3. To evaluate in vivo efficacy, Ad-hTGFbeta was intravenously injected into 7-week-old NOD mice and compared to the transduction using the vector only. The Ad-hTGFbeta group had persistent gene expression for longer than 5 weeks, and high TGFbeta serum level was secreted. There was no difference in the degree of insulitis between the Ad-hTGFbeta group and controls. Although we found favorable in vitro results, such as decrease in islet apoptosis, enhanced proliferation and differentiation, and increase in the level of CD4(+)CD25(+) regulatory T cells, there was no difference in reduction of the development of T1D between controls and Ad-hTGFbeta-injected mice. Nevertheless, if we find the appropriate mode and timing of TGFbeta gene transduction, Ad-hTGFbeta gene therapy might be useful in therapeutic cytokine delivery for the treatment of T1D.

Expression and regulation of osteoprotegerin in adipose tissue.

PURPOSE: Osteoprotegerin (OPG), a potent inhibitor of osteoclastic bone resorption, has a variety of biological functions that include anti-inflammatory effects. Adipocytes and osteoblasts share a common origin, and the formation of new blood vessels often precedes adipogenesis in developing adipose tissue microvasculature. We examined whether OPG is secreted from adipocytes, therefore contributing to the prevention of neovascularization and protecting the vessels from intimal inflammation and medial calcification. MATERIALS AND METHODS: The mRNA expression of OPG and receptor activator of NF-kappaB ligand (RANKL) was measured in differentiated 3T3L1 adipocytes and adipose tissues. RESULTS: OPG mRNA expression increased with the differentiation of 3T3L1 adipocytes, while RANKL expression was not significantly altered. OPG mRNA was expressed at higher levels in white adipose tissue than in brown adipose tissue and was most abundant in the epididymal portion. In differentiated 3T3L1 adipocytes, Rosiglitazone and insulin reduced the OPG/RANKL expression ratio in a dose- and time- dependent manner. In contrast, tumor necrosis factor-alpha (TNF-alpha) increased the expression of both OPG and RANKL in a time-dependent manner. The OPG/RANKL ratio was at a maximum two hours after TNF-alpha treatment and then returned to control levels. Furthermore, OPG was abundantly secreted into the media after transfection of OPG cDNA with Phi C31 integrase into 3T3L1 cells. CONCLUSION: Our results indicate that OPG mRNA is expressed and regulated in the adipose tissue. Considering the role of OPG in obesity-associated inflammatory changes in adipose tissue and vessels, we speculate that OPG may have both a protective function against inflammation and anti-angiogenic effects on adipose tissue.

Islet cell differentiation in liver by combinatorial expression of transcription factors neurogenin-3, BETA2, and RIPE3b1.

Transcription factors, such as PDX-1, that normally mediate pancreatic development are capable of inducing hepatic progenitor cells to differentiate into cells with pancreatic islet characteristics. We hypothesized that simultaneous expression of multiple transcription factors involved in islet development might enhance the differentiation of hepatic progenitor cells. Bi- or tri-cistronic constructs were generated in hybrid adenovirus/adeno-associated virus (Ad/AAV) vectors containing neurogenin 3 (NGN3), BETA2 (NeuroD), and RIPE3b1 (MafA), each of which plays a role in islet cell differentiation. These vectors efficiently express multiple transcription factors and stimulate insulin promoter activity in a combinatorial manner. When these multi-cistronic constructs were administered in vivo, they induce hepatic expression of islet-specific markers, including PDX-1, insulin, glucagon, somatostatin, and islet-amyloid peptide. Administration of the Ad/AAV hybrid vectors to streptozotocin-induced diabetic mice reversed hyperglycemia, consistent the differentiation of functional hepatic insulin-secreting cells. These results indicate that Ad/AAV hybrid vectors can be used to administer combinations of factors that induce islet cell differentiation in hepatic progenitor cells.

Role of the mitogen-activated protein kinases in cytokine-mediated inhibition of insulin gene expression.

BACKGROUND: Following islet transplant, inflammatory cells in the vicinity of the transplant graft elaborate cytokines that contribute to islet graft dysfunction. To better understand the mechanism for this effect of cytokines on graft function, we examined the impact of cytokines on intracellular signaling and insulin promoter activity in pancreatic beta cells. METHODS: Two pancreatic beta cell lines, RINm5F and MIN6 cells, were transfected with a rat insulin promoter (RIP) luciferase fusion gene and treated with a combination of cytokines, including 5 ng/mL interleukin-1beta + 10 ng/mL tumor necrosis factor alpha + 25 ng/mL interferon-gamma. The effect of cytokines on beta cell transcription factors and signaling pathways was analyzed by real-time reverse transcriptase polymerase chain reaction and Western blotting. RESULTS: Treatment for 48 hours with the combination of cytokines decreased insulin 1 messenger ribonucleic acid (mRNA) levels to 51% and 38% and RIP1 activity to 16% and 30% of control levels in RINm5F and MIN6 cells, respectively. The level of mRNAs encoding transcription factors important for insulin gene expression and beta cell function, including MafA, PDX-1, Nkx6.1, and Pax6, was also decreased by cytokine treatment. Cytokines increased phosphorylation of ERK and c-Jun NH2-terminal kinase (JNK) in RINm5F and MIN6 cells but had no effect on p38 kinase phosphorylation. Neither JNK nor ERK inhibition had a significant effect on cytokine-mediated inhibition of RIP1 activity. CONCLUSION: Beyond modulating beta cell survival, cytokines inhibit insulin promoter activity, which likely contributes to islet dysfunction following islet transplant. This effect appears to be mediated, in part, via altered expression of transcription factors important for insulin gene expression.

Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements.

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERalpha, a mutant ERalpha (E207A/G208A) that selectively lacks ERE binding, or ERalpha null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17beta-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERalpha signaling. This proliferative response was confirmed by 5-bromo-2'-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathway-responsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERalpha (E207A/G208A) mutant. These results indicate that nonclassical ERalpha signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERalpha plays a critical physiologic role in the uterine response to estrogen.

Gene transfer of pigment epithelium-derived factor suppresses tumor growth and angiogenesis in a hepatoblastoma xenograft model.

Normal hepatocytes express pigment epithelium-derived factor (PEDF), an endogenous antiangiogenic factor. We hypothesized that decreased PEDF expression may be one mechanism driving hepatoblastoma growth, and in vivo gene transfer of PEDF could suppress neovascularization and limit tumor growth. PEDF functional activity was determined in vitro using endothelial cell migration assays and in vivo using a subcutaneous tumor model. HUH-6 human hepatoblastoma tumors were treated with hybrid adenoviral/adeno-associated viral expression vectors for PEDF (Hyb-PEDF, n = 4) or beta-galactosidase (Hyb-betagal, n = 4) daily for 4 d. Mitotic figures, microvascular density (MVD), PEDF, and VEGF expression were assessed. Hyb-PEDF treatment inhibited in vivo tumor growth (p < 0.008) and decreased MVD (p < 0.001), the number of mitotic figures (p < 0.001), and VEGF expression when compared with Hyb-betagal-treated tumors. HUH-6 expression of PEDF was dramatically reduced when cultured under hypoxic conditions and also when grown in vivo, and the addition of neutralizing anti-PEDF antibody increased the already high baseline angiogenic activity of the HUH-6 cell secretions in vitro (p < 0.04). PEDF is an important endogenous regulator of the liver vasculature. Augmenting intra-tumoral PEDF levels inhibits tumor growth by reducing angiogenesis and VEGF expression. Potent inhibitors of angiogenesis, such as PEDF, may be an effective alternative treatment for children with hepatoblastoma.

ERE-independent ERalpha target genes differentially expressed in human breast tumors.

The classical pathway for estrogen receptor (ER) signaling is mediated by ER binding to an estrogen response element (ERE) in DNA. ERalpha can also act via a nonclassical pathway by altering the activities of other transcription factors (e.g., Sp1, AP-1, or NF-kappaB) at their cognate sites on DNA. We previously generated a mutant form of ERalpha (E207A/G208A) that does not bind to EREs, and therefore lacks signaling via the classical pathway but retains signaling via the nonclassical pathway. In the current study, we introduce this mutant ERalpha into MDA-MB231 ERalpha-negative breast carcinoma cells to identify nonclassical pathway genes that respond to 17beta-estradiol (E2), selective estrogen receptor modulators (SERMs) tamoxifen (TAM) or raloxifene (RAL), or the estrogen antagonist ICI 182,780 (ICI). Consistent with a role for nonclassical signaling in SERM action, microarray analyses identify 268 responsive nonclassical ERalpha pathway target genes. ICI elicits the largest number of nonclassical genes, followed by RAL, TAM, and E2. Custom microarrays containing identified nonclassical ERalpha responsive genes are used to compare gene expression in human breast tumor (n = 34) and normal mammary epithelial cell (n = 9) samples. A subset of nonclassical genes (n = 32) are differentially expressed in breast tumors. In summary, we show that nonclassical ERalpha pathway target genes exhibit a range of transcriptional responses to SERMs and identify targets of this pathway as potentially relevant to breast cancer. The identification of nonclassical ERalpha target genes offers new insight into estrogen receptor signaling and cross talk with pathways that mediate breast tumor response to SERM therapy.

A dominant negative peroxisome proliferator-activated receptor-gamma knock-in mouse exhibits features of the metabolic syndrome.

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARgamma mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARgamma L466A mutation, shown previously to inhibit wild-type PPARgamma action in vitro. Homozygous PPARgamma L466A knock-in mice die in utero. Heterozygous PPARgamma L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARgamma function and the metabolic syndrome.

Peroxisome proliferator-activated receptor gamma agonists promote TRAIL-induced apoptosis by reducing survivin levels via cyclin D3 repression and cell cycle arrest.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrate that human breast cancer cells, but not normal mammary epithelial cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione (TZD) class. Although TZDs do not significantly alter the expression of components of the TRAIL signaling pathway, they profoundly reduce protein levels of cyclin D3, but not other D-type cyclins, by decreasing cyclin D3 mRNA levels and by inducing its proteasomal degradation. Importantly, both TRAIL sensitization and reduction in cyclin D3 protein levels induced by TZDs are likely PPARgamma-independent because a dominant negative mutant of PPARgamma did not antagonize these effects of TZDs, nor were they affected by the expression levels of PPARgamma. TZDs also inhibit G(1) to S cell cycle progression. Furthermore, silencing cyclin D3 by RNA interference inhibits S phase entry and sensitizes breast cancer cells to TRAIL, indicating a key role for cyclin D3 repression in these events. G(1) cell cycle arrest sensitizes breast cancer cells to TRAIL at least in part by reducing levels of the anti-apoptotic protein survivin: ectopic expression of survivin partially suppresses apoptosis induced by TRAIL and TZDs. We also demonstrate for the first time that TZDs promote TRAIL-induced apoptosis of breast cancer in vivo, suggesting that this combination may be an effective therapy for cancer.

Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: role of AF-1 and AF-2.

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection. To examine the role of the transcriptional activation function (AF) domains of ERalpha in resveratrol agonism, we compared the effects of resveratrol and estradiol (E2) on expression of exogenous reporter genes and an endogenous estrogen-regulated gene (TGFalpha) in MDA-MB-231 cells stably transfected with wild-type (wt) ERalpha or mutants with deleted or mutated AF domains. In reporter gene assays, cells expressing wtERalpha showed a superagonistic response to resveratrol. Deletion of AF-1 or mutation of AF-2 attenuated the effect of resveratrol disproportionately compared to that of E2, while deletion of AF-2 abrogated the response to both ligands. In TGFalpha expression assays, resveratrol acted as a full agonist in cells expressing wtERalpha. Deletion of AF-1 attenuated stimulation by E2 more severely than that by resveratrol, as did deletion of AF-2. In contrast, mutation of AF-2 left both ligands with a limited ability to induced TGFalpha expression. In summary, the effect of modifying or deleting AF domains depends strongly on the ligand and the target gene.