Independent differentiation of mammotropes and somatotropes in the chicken embryonic pituitary gland

It has been reported that mammotropes in a rodent pituitary gland are derived from somatotropes via somatomammotropes (SMTs), cells that produce both growth hormone (GH) and prolactin (Prl). However, no studies have been done on the transdifferentiation of somatotropes in the chicken pituitary gland. In this study, in order to determine the origin of mammotropes, we studied detail property of appearance of chicken somatotropes, mammotropes and pit-1 cells and then evaluated the existence of SMTs in the chicken embryonic pituitary gland. Immunohistochemical analysis revealed that GH-immunopositive (GH-ip) cells appeared on embryonic day (E) 14 and were mainly distributed in the caudal lobe, while Prl-immunopositive (Prl-ip) cells appeared in the cephalic lobe of the pituitary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and Prl mRNA starts at E12 in the caudal lobe and at E14 in the cephalic lobe respectively. Pit-1 mRNA was first detected on E5 by RT-PCR, and pit-1 mRNA-expressing cells were found in the cephalic lobe on E8. Then with the ontogeny of the chicken, these cells spread into both lobes. Using a double staining method with ISH and immunohistochemistry, we could not detect the existence of SMTs in the chicken embryonic pituitary gland even in the marginal region of either lobe. These results suggest that chicken somatotropes and mammotropes independently appear in different lobes of pituitary gland and that transdifferentiation from somatotropes to mammotropes is not the central route for differentiation of mammotropes in the embryonic chicken pituitary gland.     

Correlation between TGF-β2/3 promoter DNA methylation and Smad signaling during palatal fusion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant that is strongly associated with a number of human diseases and birth defects, including cleft palate. Transforming growth factor (TGF) plays a significant role during mammalian palatogenesis. However, the epigenetic mechanism of transforming growth factors in the process of TCDD-induced cleft palate is unclear. The purpose of this research was to investigate the relationship and potential mechanism between TGF-β2/3 promoter DNA methylation and Smad signaling during TCDD-induced cleft palate. Pregnant C57BL/6N mice were exposed to 64 µg/kg TCDD on gestational day 10 (GD10) to establish the cleft palate model and palatal tissues of embryos were collected on GD13, GD14, and GD15 for subsequent experiments. TGF-β2/3 mRNA expression, TGF-β2/3 promoter methylation, and Smad signaling molecules expression were assessed in the palate of the two groups. The results showed that the incidence of cleft palate was 94.7% in the TCDD-treated group whereas no cleft palate was found in the control group. TCDD-treated group altered specific CpG sites of TGF-β2/3 promoter methylation. Compared to the control group, the proliferation of mouse embryonic palate mesenchymal stromal cells (MEPM), the expressions of TGF-β2/3, p-Smad2, and Smad4 were all reduced, while the expression of Smad7 was significantly increased in the atAR group. Smad signaling was downregulated by TCDD. Therefore, we suggest that TGF-β2/3 promoter methylation and Smad signaling may be involved in TCDD-induced cleft palate formation in fetal mice.     

Proteomic and histochemical analysis of proteins involved in the dying-back-type of axonal degeneration in the gracile axonal dystrophy (gad) mouse

Local axonal degeneration is a common pathological feature of peripheral neuropathies and neurodegenerative disorders of the central nervous system, including Alzheimer's disease, Parkinson's disease, and stroke; however, the underlying molecular mechanism is not known. Here, we analyzed the gracile axonal dystrophy (gad) mouse, which displays the dying-back-type of axonal degeneration in sensory neurons, to find the molecules involved in the mechanism of axonal degeneration. The gad mouse is analogous to a null mutant of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). UCH-L1 is a deubiquitinating enzyme expressed at high levels in neurons, as well as testis and ovary. In addition, we recently discovered a new function of UCH-L1—namely to bind to and stabilize mono-ubiquitin in neurons, and found that the level of mono-ubiquitin was decreased in neurons, especially in axons of the sciatic nerve, in gad mice. The low level of ubiquitin suggests that the target proteins of the ubiquitin proteasome system are not sufficiently ubiquitinated and thus degraded in the gad mouse; therefore, these proteins may be the key molecules involved in axonal degeneration. To identify molecules involved in axonal degeneration in gad mice, we compared protein expression in sciatic nerves between gad and wild-type mice at 2 and 12 weeks old, using two-dimensional difference gel electrophoresis. As a result, we found age-dependent accumulation of several proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 14-3-3, in gad mice compared with wild-type mice. Histochemical analyses demonstrated that GAPDH and 14-3-3 were localized throughout axons in both gad and wild-type mice, but GAPDH accumulated in the axons of gad mice. Recently, it has been suggested that a wide range of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates, and it has been reported that oxidative stress causes the aggregation of GAPDH. Furthermore, histochemical analysis demonstrated that sulfonated GAPDH, a sensor of oxidative stress that elicits cellular dysfunction, was expressed in the axons of gad mice, and 4-hydroxy-2-nonenal, a major marker of oxidative stress, was also only detected in gad mice. Our findings suggest that GAPDH may participate in a process of the dying-back-type of axonal degeneration in gad mice and may provide valuable insight into the mechanisms of axonal degeneration.     

Deficiency of Neuronal p38α MAPK Attenuates Amyloid Pathology in Alzheimer Disease Mouse and Cell Models through Facilitating Lysosomal Degradation of BACE1

Amyloid β (Aβ) damages neurons and triggers microglial inflammatory activation in the Alzheimer disease (AD) brain. BACE1 is the primary enzyme in Aβ generation. Neuroinflammation potentially up-regulates BACE1 expression and increases Aβ production. In Alzheimer amyloid precursor protein-transgenic mice and SH-SY5Y cell models, we specifically knocked out or knocked down gene expression of mapk14, which encodes p38α MAPK, a kinase sensitive to inflammatory and oxidative stimuli. Using immunological and biochemical methods, we observed that reduction of p38α MAPK expression facilitated the lysosomal degradation of BACE1, decreased BACE1 protein and activity, and subsequently attenuated Aβ generation in the AD mouse brain. Inhibition of p38α MAPK also enhanced autophagy. Blocking autophagy by treating cells with 3-methyladenine or overexpressing dominant-negative ATG5 abolished the deficiency of the p38α MAPK-induced BACE1 protein reduction in cultured cells. Thus, our study demonstrates that p38α MAPK plays a critical role in the regulation of BACE1 degradation and Aβ generation in AD pathogenesis.     

UBE2C triggers HIF‐1α‐glycolytic flux in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy in Taiwan. Therefore, refining the diagnostic sensitivity of biomarkers for early‐stage tumours and identifying therapeutic targets are critical for improving the survival rate of HNSCC patients. Metabolic reprogramming contributes to cancer development and progression. Metabolic pathways, specifically, play a crucial role in these diverse biological and pathological processes, which include cell proliferation, differentiation, apoptosis and carcinogenesis. Here, we investigated the role and potential prognostic value of the ubiquitin‐conjugating enzyme E2 (UBE2) family in HNSCC. Gene expression database analysis followed by tumour comparison with non‐tumour tissue showed that UBE2C was upregulated in tumours and was associated with lymph node metastasis in HNSCC patients. Knockdown of UBE2C significantly reduced the invasion/migration abilities of SAS and CAL27 cells. UBE2C modulates glycolysis pathway activation and HIF‐1α expression in SAS and CAL27 cells. CoCl₂ (HIF‐1α inducer) treatment restored the expression of glycolytic enzymes and the migration/invasion abilities of UBE2C knockdown cells. Based on our findings, UBE2C expression mediates HIF‐1α activation, increasing glycolysis pathway activation and the invasion/migration abilities of cancer cells. UBE2C may be an independent prognostic factor and a therapeutic target in HNSCC.     

Ubiquitin dimers control the hydrolase activity of UCH-L3

Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.     

Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic β cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F₁ mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F₁ mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F₁ fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F₁ mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic β cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.     

Cideb facilitates the lipidation of chylomicrons in the small intestine

Cell death-inducing DFF45-like effector b (Cideb), an endoplasmic reticulum (ER)- and lipid droplet (LD)-associated protein, has been shown to play a critical role in maintaining hepatic lipid homeostasis by promoting the lipidation and maturation of VLDL particles. Here, we observed that Cideb is expressed in the jejunum and ileum sections of the small intestine, and its expression was induced by high-fat diet. Intragastric gavage with lipids resulted in the retention of lipids in the intestine in Cideb-deficient mice. In addition, we observed that mice with Cideb deficiency exhibited reduced intestinal chylomicron-TG secretion and increased lipid accumulation in the enterocytes. The sizes of chylomicrons secreted from the small intestine of Cideb-deficient mice were also smaller than those from wild-type mice. Furthermore, the overexpression of Cideb increased TG secretion and reduced lipid accumulation in the enterocyte-like Caco-2 cells. In addition, we proved that Cideb was localized to the ER and LDs and could interact with ApoB48 in Caco-2 cells. Overall, these data revealed that Cideb plays an important role in controlling intestinal chylomicron lipidation.     

Wild-type IDH1 inhibits the tumor growth through degrading HIF-α in renal cell carcinoma

The purpose of our study was to explore the effect and intrinsic mechanism of wild-type IDH1 and its substrate α-KG on renal cell carcinoma (RCC). IDH1 was observed lower expression in RCC cell lines. Phenotype experiment was carried out in the wild-type IDH1 and mutant IDH1^R132H plasmid treated cell line. The results showed that the wild-type IDH1 could significantly inhibit the proliferation, migration and promote the apoptosis of RCC cell lines, which were consistent with the IDH1''s substrate α-KG. The mutant IDH1^R132H was found to lose this biological function of IDH1. Moreover, we verified the proliferation inhibition of IDH1 in vivo. In addition, we verified the correlation between IDH1 and hypoxia signal-related proteins in vitro and in vivo, specifically, IDH1 overexpression could significantly reduce the expression of HIF-1α and HIF-2α proteins and its downstream proteins (VEGF, TGF-α). Furthermore, we preliminarily verified the possibility of α-KG in the RCC''s treatment by injecting α-KG into the xenograft model. α-KG significantly reduced tumor size and weight in tumor-bearing mice. This study provided a new therapeutic target and small molecule for the study of the treatment and mechanism of RCC.     

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein β-catenin. Jak3 expression in these cells was essential for AJ localization of β-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of β-catenin.     

TNF-α Suppresses Apelin Receptor Expression in Mouse Quadriceps Femoris-Derived Cells

Expression of the apelin receptor, APJ, in skeletal muscle (SM) is known to decrease with age, but the underlying mechanism remains unclear. Increased tumor necrosis factor (TNF)-α levels are observed in SM with age and are associated with muscle atrophy. To investigate the possible interconnection between TNF-α elevation and APJ reduction with aging, we investigated the effect of TNF-α on APJ expression in cells derived from the quadriceps femoris of C57BL/6J mice. Expression of Tnfa and Apj in the quadriceps femoris was compared between 4- (young) and 24-month-old (old) C57BL/6J mice (n = 10 each) using qPCR. Additionally, APJ-positive cells and TNF-α protein were analyzed by flow cytometry and Western blotting, respectively. Further, quadricep-derived cells were exposed to 0 (control) or 25 ng/mL TNF-α, and the effect on Apj expression was examined by qRT-PCR. Apj expression and the ratio of APJ-positive cells among quadricep cells were significantly lower in old compared to young mice. In contrast, levels of Tnfa mRNA and TNF-α protein were significantly elevated in old compared to young mice. Exposing young and old derived quadricep cells to TNF-α for 8 and 24 h caused Apj levels to significantly decrease. TNF-α suppresses APJ expression in muscle cells in vitro. The increase in TNF-α observed in SM with age may induce a decrease in APJ expression.     

IRF2 enhances RANKL-induced osteoclast differentiation via regulating NF-κB/NFATc1 signaling

Interferon regulatory factors (IRFs) play roles in various biological processes including cytokine signaling, cell growth regulation and hematopoietic development. Although it has been reported that several IRFs are involved in bone metabolism, the role of IRF2 in bone cells has not been elucidated. Here, we investigated the involvement of IRF2 in RANKL-induced osteoclast differentiation. IRF2 overexpression in osteoclast pre-cursor cells enhanced osteoclast differentiation by regulating the expression of NFATc1, a master regulator of osteoclasto-genesis. Conversely, IRF2 knockdown inhibited osteoclast differentiation and decreased the NFATc1 expression. Moreover, IRF2 increased the translocation of NF-κB subunit p65 to the nucleus in response to RANKL and subsequently induced the expression of NFATc1. IRF2 plays an important role in RANKL-induced osteoclast differentiation by regulating NF-κB/NFATc1 signaling pathway. Taken together, we demonstrated the molecular mechanism of IRF2 in osteoclast differentiation, and provide a molecular basis for potential therapeutic targets for the treatment of bone diseases characterized by excessive bone resorption.