Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

1α,25‐dihydroxyvitamin D3 acts via transforming growth factor‐β to up‐regulate expression of immunosuppressive CD73 on human CD4+ Foxp3– T cells

Vitamin D deficiency is associated with increased incidence and severity of various immune‐mediated diseases. Active vitamin D (1α,25‐dihydroxyvitamin D3; 1,25(OH)₂D3) up‐regulates CD4⁺ T‐cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti‐inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)₂D3 on expression of the downstream ecto‐5′‐nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor‐β (TGF‐β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10^?8 to 10^?7 m , 1,25(OH)₂D3 significantly increased expression of CD73 on peripheral human CD4⁺ T cells. Although 1,25(OH)₂D3 did not affect the mRNA expression of latent TGF‐β₁, 1,25(OH)₂D3 did up‐regulate expression of TGF‐β‐associated molecules [latency‐associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin‐1, thrombospondin‐1 and α_v integrin] which is likely to have contributed to the observed enhancement in TGF‐β bioactivity. CD73 was highly co‐expressed with LAP and GARP following 1,25(OH)₂D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4⁺ Foxp3^– T cells, rather than CD4⁺ Foxp3⁺ T cells. Notably, neutralization of TGF‐β significantly impaired 1,25(OH)₂D3‐mediated induction of CD73. Collectively, we show that 1,25(OH)₂D3 enhances expression of CD73 on CD4⁺ Foxp3^– T cells in a process that is at least partially TGF‐β‐dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune‐mediated disease.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.     

Reduced expression of oestrogen receptor‐β is associated with tumour invasion and metastasis in oestrogen receptor‐α‐negative human papillary thyroid carcinoma

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)‐α and ‐β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα‐negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild‐type ERβ) and its clinical significance in female ERα‐negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα‐negative PTC patients. The total score for ERβ1 was significantly lower in female ERα‐negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = ?2.923, P = 0.003 and Z = ?3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα‐negative PTC patients expressing E‐cadherin compared to patients negative for E‐cadherin expression (Z = ?2.636, P = 0.008). The total score was lower in ERα‐negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = ?1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα‐negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease.     

The stimulatory effect of α‐melanotropin on progesterone release from rat granulosa cells is inhibited by interleukin‐1β and by tumour necrosis factor‐α

Aim: Several studies have shown that a variety of peptides and cytokines are involved in ovarian regulatory mechanisms; however, their exact function is still unclear. In this work we study whether the administration of peptide α‐melanotropin and the cytokines interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) on their own modify the release of progesterone in cultured granulosa cells (GC) from pro‐oestrous rats. We also investigate an interaction between these cytokines and α‐melanotropin in the modulation of progesterone secretion. Methods: Granulosa cells were collected from the ovaries of female Wistar rats and cultured for up to 24 h in the presence of different concentrations of α‐melanotropin, cytokines or a combination of both. Progesterone concentration was measured by radioimmunoassay. Results: The addition of α‐melanotropin in a dose of 0.01 and 0.1 mm had no effect on progesterone release, whereas a dose of 1 mm significantly increased progesterone release (P < 0.01) compared with the control culture. Progesterone release was not modified when different concentrations of interleukin‐1β or TNF‐α were added to the cell cultures. However, when interleukin‐1β or TNF‐α were added simultaneously with 1 μm α‐melanotropin, a significant reduction (P < 0.01 for interleukin‐1β and P < 0.05 for TNF‐α) of the steroid release was found with respect to the α‐melanotropin‐treated group. Conclusions: These results lead us to suggest that, although α‐melanotropin stimulates progesterone release in pre‐ovulatory GC, this effect is blocked by the presence of interleukin‐1β or TNF‐α.     

Berbamine Enhances the Antineoplastic Activity of Gemcitabine in Pancreatic Cancer Cells by Activating Transforming Growth Factor‐β/Smad Signaling

Drug‐resistance to gemcitabine chemotherapy in pancreatic cancer is still an unsolved problem. Combinations of other chemotherapy drugs with gemcitabine have been shown to increase the efficacy of gemcitabine‐based treatment. In this study, the effect of berbamine on the antitumor activity of gemcitabine was evaluated in human pancreatic cancer cell lines Bxpc‐3 and Panc‐1, and the underlying mechanisms were explored. Our results demonstrated that berbamine exhibited a time‐ and dose‐dependent inhibitory effect in the pancreatic cancer cell lines. Berbamine enhanced gemcitabine‐induced cell growth inhibition and apoptosis in these cells. Combined treatment of berbamine and gemcitabine resulted in down‐regulation of anti‐apoptotic proteins (Bcl‐2, Bcl‐xL) and up‐regulation of pro‐apoptotic proteins (Bax, Bid). More importantly, berbamine treatment in combination with gemcitabine activated the transforming growth factor‐β/Smad (TGF‐β/Smad) signaling pathway, as a result of a decrease in Smad7 and an increase in transforming growth factor‐β receptor II (TβRII) expression. Changes in downstream targets of Smad7, such as up‐regulation of p21 and down‐regulation of c‐Myc and Cyclin D1 were also observed. Therefore, berbamine could enhance the antitumor activity of gemcitabine by inhibiting cell growth and inducing apoptosis, possibly through the regulation of the expression of apoptosis‐related proteins and the activation of TGF‐β/Smad signaling pathway. Our study indicates that berbamine may be a promising candidate to be used in combination with gemcitabine for pancreatic cancer treatment. Anat Rec, 297:802–809, 2014. © 2014 Wiley Periodicals, Inc.     

Impaired Cytoplasmic–Nuclear Transport of Hypoxia‐Inducible Factor‐1α in Amyotrophic Lateral Sclerosis

We investigated the mechanisms underlying abnormal vascular endothelial growth factor (VEGF) production in amyotrophic lateral sclerosis (ALS). We immunohistochemically studied VEGF, its receptors VEGFR1 and 2, and hypoxia‐inducible factor‐1α (HIF‐1α) in autopsied ALS spinal cords. We also chronologically assessed the expression of HIF‐1α, karyopherin β1, karyopherin β‐cargo protein complex inhibitors and nuclear pore complex proteins in G93A mutant superoxide dismutase 1 (mSOD1) transgenic mice at presymptomatic, symptomatic and end stages. In ALS patients, compared with controls, HIF‐1α immunoreactivity in the cytoplasm of anterior horn cells (AHCs) was significantly increased, while immunoreactivities for VEGF and VEGFRs were significantly decreased. Similar changes in HIF‐1α and VEGF levels were observed in mSOD1 transgenic mice. HIF‐1α co‐localized with karyopherin β1 in the cytoplasm of AHCs and karyopherin β1 co‐localized with nucleoporin 62 (Nup62) on the nuclear envelope. From the presymptomatic stage of mSOD1 transgenic mice, karyopherin β1 immunoreactivity in AHC nuclei significantly decreased and morphological irregularities of the Nup62‐immunostained nuclear envelope became more pronounced with disease progression. Thus, in AHCs from mSOD1 transgenic mice, transport of cytoplasmic HIF‐1α to the nuclear envelope and into the nucleus is impaired from the presymptomatic stage, suggesting that impaired cytoplasmic–nuclear transport of HIF‐1α through the nuclear pore might precede motor neuron degeneration.     

Muscleblind‐like 1 is a negative regulator of TGF‐β‐dependent epithelial–mesenchymal transition of atrioventricular canal endocardial cells

The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind‐like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor β‐dependent increase in epithelial–mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development. Developmental Dynamics 238:3266–3272, 2009. © 2009 Wiley‐Liss, Inc.     

Hypoxic inactivation of glycogen synthase kinase‐3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia‐inducible factor‐1 signaling

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell‐type specific, we investigated whether glycogen synthase kinase‐3β (GSK‐3β) activation is involved in hypoxia‐induced gastric tumor promotion. Stable gastric cancer cell lines (SNU‐638, SNU‐484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild‐type GSK‐3β (WT‐GSK‐3β) or kinase‐dead mutant of GSK‐3β (KD‐GSK‐3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK‐3β activation in gastric cancer cells. Cell viability and the expressions of HIF‐1α protein and VEGF mRNA in gastric cancer cells were higher in KD‐GSK‐3β transfectants than in WT‐GSK‐3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF‐1α activation, and VEGF expression were higher in KD‐GSK‐3β tumors than in WT‐GSK‐3β tumors in vivo. In addition, the expression of hypoxia‐induced HIF‐1α protein was regulated by GSK‐3β at the translational level. Our data suggest that GSK‐3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF‐1α/VEGF signaling pathway.     

Transforming growth factor-β1-induced expression of the mucosa-related integrin αE on lymphocytes is not associated with mucosa-specific homing

The integrin α_E (HML-1, α_IEL, α_M290) is largely expressed on lymphocytes in epithelial sites, especially the gut mucosa. We investigated whether α_E has any role in homing or delineates a phenotype with distinct migratory behavior. Lymph node T cells were stimulated for 5 days with anti-CD3 in the presence or absence of transforming growth factor (TGF)-β₁ to generate α_E⁺ or α_E^? cells, respectively. The two populations were then tested for their homing properties in mice. Both α_E⁺ (TGF-β-treated) and α_E^? (control) cells of either CD4⁺ or CD8⁺ subset had a low capacity to enter the gut and showed the same homing behavior with respect to a variety of other organs. The same was true for α_E⁺ and α_E^? cells that had been briefly stimulated with anti-CD3 (24 h) and then allowed to return to a resting state before injection, though in this case both populations showed a greater capacity to recirculate through lymphoid tissue than was seen with fully activated cells. The results indicate that α_Eβ₇ does not act as a homing receptor, and that the expression of the site-specific marker α_E does not correlate with a distinct homing behavior.     

Changes in calsequestrin,TNF‐α, TGF‐β and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps,diaphragm and intrinsic laryngeal muscles

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium‐related protein), tumour necrosis factor (TNF‐α; pro‐inflammatory cytokine), tumour growth factor (TGF‐β; pro‐fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF‐α, TGF‐β and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF‐α and TGF‐β serve as markers of dystrophy primarily for the diaphragm.     

Epigallocatechin‐3‐Gallate Inhibits Ethanol‐Induced Apoptosis Through Neurod1 Regulating CHOP Expression in Pancreatic β‐Cells

Epiga‐llocatechin‐3‐gallate (EGCG) is one kind of polyphenol abundant extracted from green tea which has a potent antidiabetic activity. However, the molecular mechanisms mediating the protection procession of EGCG are still unclear. The aim of this study was to investigate the protective effect of EGCG on pancreatic β‐cells exposed to ethanol and the possible underlying mechanisms. To observe the effect of EGCG, we assessed apoptosis in βTC‐6 and INS‐1 cells, which were in complete medium containing 60 mM ethanol, or coincubation with different concentration of EGCG. We also evaluated the roles of Neurod1 in CHOP expression and ethanol‐mediated damage through plasmid overexpression. Treatment with EGCG decreased CHOP expression and apoptosis, whereas its treatment increased Neurod1 expression in ethanol‐treated βTC‐6 and INS‐1 cells. Overexpression of Neurod1 caused the decrease of CHOP expression and apoptosis in ethanol‐treated cells. Furthermore, Neurod1 inhibited CHOP expression by deacetylation of Histone H4 at the CHOP gene promoter. In addition, EGCG partially restores the activity of Neurod1 binding to CHOP promoter in ethanol‐treated cells. In conclusion, EGCG protected β‐cell against ethanol‐induced β‐cell apoptosis by Neurod1 regulating CHOP expression. Anat Rec, 299:573–582, 2016. © 2016 Wiley Periodicals, Inc.