Angiotensin II directly impairs adipogenic differentiation of human preadipose cells

microRNAs (miRNAs) are small non-coding RNAs that have been suggested to play an essential role in tumorigenesis. Reduced expression of miR-338 has been reported in several types of cancers; however, the role of miR-338 in oral squamous cell carcinoma (OSCC) has not been elucidated. In this study, we demonstrated that miR-338 was dramatically downregulated in OSCC tissues and cell lines. Overexpression of miR-338 significantly inhibited proliferation, colony formation, migration, and invasion of OSCC cells. In addition, neuropilin1 (NRP1) was identified as a target of miR-338 in OSCC cells and inversely correlated with miR-338 in OSCC tissues. Furthermore, restoration of NRP1 attenuated the tumor-suppressive effects of miR-338. Taken together, miR-338 might inhibit growth and metastasis of OSCC cells by targeting NRP1.     

Molecular characterization of angiotensin II type II receptors in rat pheochromocytoma cells.

The binding sites and biochemical effects of angiotensin (A) II were investigated in rat pheochromocytoma (PC12W) cells. Sarcosine1, [125I]-tyrosine4, isoleucine8-AII ([125I]-SI-AII) bound to a saturable population of sites on membranes with an equilibrium dissociation constant (Kd) of 0.4 nM and a binding site maximum of 254 fmol/mg protein. Competitive displacement of [125I]-SI-AII by agonists and antagonists elucidated a rank order of potency of AIII greater than or equal to AII greater than PD 123177 greater than AI greater than [des-Phe]AII [AII(1-7)] much greater than DuP 753. The stable guanine nucleotide analog 5'-guanylyl imidodiphosphate did not alter the binding affinity or slope of the inhibition curves for AI, AII, AIII, or AII(1-7). Treatment of PC12W cells with AII or AIII did not affect the free intracellular calcium concentration, phosphoinositide metabolism, arachidonate release, cyclic GMP, or cyclic AMP concentrations. [125I]-AII binding sites remained on the cell surface and were not internalized after 2 h at 37 degrees C. Angiotensin II did not stimulate tyrosine, serine, or threonine phosphorylation. Northern analysis of PC12W mRNA with an AT1 receptor gene probe failed to produce an RNA:DNA hybrid at low stringency. These data indicate that PC12W cells express a homogeneous population of AT2 binding sites which differ significantly from AT1 receptors in signal transduction and molecular structure. AT2 sites may act via potentially novel, biochemical pathways or, alternatively, be vestigial receptors.     

Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.     

Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing.

We localized and characterized angiotensin II AT1 and AT2 receptors in the skin of 2-week-old rats during experimental wound healing. Both AT1 and AT2 were present in the skin. Three days after wounding, the expression of angiotensin II receptors was significantly enhanced in the dermis as well as in a localized band within the superficial dermis of the skin surrounding the wound. The major proportion of this increase was due to angiotensin II AT2 receptors. Our results suggest a physiological role for AT2 receptors in the process of tissue repair.     

Expression of the H type 1 blood group antigen during enterocytic differentiation of Caco-2 cells.

We made a comparative study of the structures of the oligosaccharides on the glycoproteins from Caco-2 human colonic adenocarcinoma cells, before and after differentiation. Enterocytic differentiated Caco-2 cells highly express H type 1 blood group antigen on the cell surface as well as activities of brush border membrane hydrolases, such as dipeptidyl peptidase IV and alkaline phosphatase. A strong correlation was observed between the amounts of H type 1 blood group antigen and the degrees of differentiation. Structural analysis with use of lectin affinity high performance liquid chromatography revealed that typical mucin-type sugar chains of the glycoproteins from undifferentiated cells have H type 2 group, linear polylactosamines, and core 1 structure. On the other hand, differentiated cells newly contain H type 1 and Le(b) groups and core 2 structure. Mucins with H type 1 group make contact with brush border membrane enzymes on differentiated cells. Furthermore benzyl 2-acetamide-2-deoxy-alpha-D-galactopyranoside inhibited both expression of H type 1 group on the cell surface and enhancement of brush border membrane enzyme activities even in the presence of a differentiating inducer. These results suggest that the mucin-type sugar chains with H type 1 group have important functions regarding differentiation of Caco-2 cells.     

Larger anti-adipogenic effect of angiotensin II on omental preadipose cells of obese humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans. Research Methods and Procedures: The angiotensin II effect on adipose cell formation was evaluated in human omental adipocyte progenitor cells that were stimulated to adipogenic differentiation in vitro. The adipogenic response was measured by the activity of the differentiation marker glycerol‐3‐phosphate dehydrogenase. Results: Angiotensin II reduced the adipogenic response of adipocyte progenitor cells, and the extent of the decrease correlated directly with the subjects’ BMI (p = 0.01, R² = 0.30). A 56.3 ± 3.4% and 44.5 ± 2.7% reduction of adipogenesis was found in obese and non‐obese donors’ cells, respectively (p < 0.01). The effect of angiotensin II was reversed by type 1 angiotensin receptor antagonist losartan. Discussion: A greater anti‐adipogenic response to angiotensin II in omental adipose progenitor cells from obese subjects opens a venue to understand the deregulation of visceral fat tissue cellularity that has been associated with severe functional abnormalities of the obese condition.     

Interaction between bradykinin subtype 2 and angiotensin II type 2 receptors during post-MI left ventricular remodeling

Angiotensin II type 2 receptor (AT(2)R) overexpression (AT(2)TG) attenuates left ventricular remodeling in a mouse model of anterior myocardial infarction (MI). We hypothesized that the beneficial effects of cardiac AT(2)TG are mediated via the bradykinin subtype 2 receptor (B(2)R). Fourteen transgenic mice overexpressing the AT(2)R (AT(2)TG mice), 10 mice with a B(2)R deletion (B(2)KO mice), 13 AT(2)TG mice with B(2)R deletion (AT(2)TG/B(2)KO mice), and 11 wild-type (WT) mice were studied. All mice were on a C57BL/6 background. Mice were studied by cardiac magnetic resonance imaging at baseline and days 1, 7, and 28 after MI induced by 1 h of occlusion of the left anterior descending artery followed by reperfusion. Short-axis images from apex to base were used to compare ventricular volumes and ejection fraction (EF). At baseline, end-diastolic volume index (EDVI) and end-systolic volume index (ESVI) were lower and EF higher in AT(2)TG mice compared with the other three strains. Infarct size was similar between groups. No differences were observed in global remodeling parameters at day 28 between AT(2)TG and AT(2)TG/B(2)KO mice; however, EDVI and ESVI were lower and EF higher in both transgenic groups than in WT or B(2)KO mice. Both strains lacking B(2)R demonstrated increased collagen content and less hypertrophy in adjacent noninfarcted regions at day 28. Attenuation of postinfarct remodeling by overexpression of AT(2)R is not directly mediated via a B(2)R pathway. However, B(2)R does appear to have a role in the smaller cavity size and hyperdynamic function observed at baseline in AT(2)TG mice and in limiting collagen deposition during postinfarct remodeling.     

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.     

Renal effects of angiotensin II in the newborn period: role of type 1 and type 2 receptors

Background

Evidence suggests a critical role for the renin-angiotensin system in regulating renal function during postnatal development. However, the physiological relevance of a highly elevated renin-angiotensin system early in life is not well understood, nor which angiotensin receptors might be involved. This study was designed to investigate the roles of angiotensin receptors type 1 (AT1R) and type 2 (AT2R) in regulating glomerular and tubular function during postnatal development.

Methods

The renal effects of the selective antagonist to AT1R, ZD 7155 and to AT2R, PD 1233319 were evaluated in two groups of conscious chronically instrumented lambs aged?~?one week (N?=?8) and?~?six weeks (N?=?10). Two experiments were carried out in each animal and consisted of the assessment of renal variables including glomerular and tubular function, for 30 min before (Control) and 60 min after infusion of ZD 7155 and PD 123319, respectively. Statistical significance was determined using parametric testing (Student t-test, analysis of variance ANOVA) as appropriate.

Results

ZD 7155 infusion was associated with a significant decrease in glomerular filtration rate and filtration fraction at one but not six weeks; urinary flow rate decreased significantly in older animals, whereas sodium excretion and free water clearance were not altered. There was an age-dependent effect on potassium handling along the nephron, potassium excretion decreasing after ZD 7155 infusion in younger but not in older lambs. PD 123319 had no significant effects on glomerular filtration rate and tubular function in either age group.

Conclusions

These results provide evidence to support an important role for AT1Rs in mediating the renal effects of angiotensin II during postnatal maturation in conscious developing animals. In contrast to a role for AT2Rs later in life, there appears to be no role for AT2Rs in influencing the renal effects of Angiotensin II in the postnatal period.

     

Expression of estrogen receptors during growth of human pancreatic adenocarcinoma cells (Capan-1)-relationship with differentiation

Summary In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic duct cells, the expression of estrogen receptor (ERα) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1 cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERα and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERα (236 ± 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%). At the onset of the stationary phase (Day 5), ERα levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 ± 0.9 fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen. Immunocytochemical analysis with anti-ERα-1D5 antibodies showed nuclear and cytoplasmic localization of ERα in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERα is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in nontarget tissues.     

Expression of angiotensin type 1 and 2 receptors in brain after transient middle cerebral artery occlusion in rats

Angiotensin II (Ang II) type 2 receptors (AT2Rs) have been associated with apoptosis. We hypothesized that AT2Rs are increased in stroke and may contribute effects of stroke to the brain. To test this, we have examined the expression of Ang II type 1 receptor (AT1R), AT2R and Ang II levels in the brain 24 h after transient middle cerebral artery occlusion (MCAO). The densities of AT1R and AT2R were measured by quantitative autoradiography (n=6). The levels of Ang II were measured by radioimmunoassay (RIA) (n=6) and by immunohistochemistry (n=3). AT1R levels on autoradiography showed a significant decrease (0.87±0.06 to 1.39±0.07 fmol/mg, p<0.01) in the ventral cortex of the stroke side compared to the cortices of non-stroke (NS) rats (n=4). There was no significant difference on ATIR in the contralateral verbal cortex of the stroke rats compared to NS control. In contrast, levels of AT2R in the ventral cortex of both the stroke and the contralateral sides were significantly increased (0.77±0.06, p<0.05 and 0.91±0.05, p<0.01 compared to 0.60±0.03 fmol/mg tissue, respectively). RIA showed that Ang II in the ventral cortex of both the stroke and the contralateral sides were significantly increased (241.63±47.72, p<0.01 and 165.51±42.59, p<0.05 compared to 76.80±4.10 pg/g tissue, respectively). Also, Ang II in the hypothalamus was significantly increased (179.50±17.49 to 118.50±6.65 pg/g tissue, p<0.05). Immunohistochemistry confirmed the increase of Ang II. These results demonstrate that brain Ang II and AT2Rs are increased whereas AT1Rs are decreased after transient MCAO in rats. We conclude that in stroke, Ang II and AT2R are activated and may contribute neural effects to brain ischemia.     

Expression of retinoid receptors during the retinoic acid-induced neuronal differentiation of human embryonal carcinoma cells

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.     

Selective type 1 angiotensin II receptor blockade attenuates oxidative stress and regulates angiotensin II receptors in the canine failing heart

Background and objective Angiotensin II type 1 receptor (AT₁R) blockade reduces vascular oxidative stress but whether myocardial oxidative stress represents a mechanism for the beneficial effect of AT₁R blockade in heart failure is unclear. Furthermore, the impact of AT₁R blockade on the expression of angiotensin II receptors in heart failure has not been well documented. Accordingly, we examined the impact of the AT₁R blocker candesartan on hemodynamics, left ventricular (LV) remodeling (echocardiography), oxidative stress, and tissue expression of AT₁Rs and angiotensin II type 2 receptors (AT₂Rs) in a canine model of pacing-induced heart failure. Methods and results Animals were randomized to rapid right ventricular-pacing (250 beats/min for 3 weeks) to severe heart failure and treated with candesartan (10 mg/kg daily, n = 8) or placebo (n = 8) from day 3 onwards, or no pacing (sham, n = 7). Candesartan significantly reduced mean pulmonary arterial and LV diastolic pressure, LV end-diastolic and end-systolic volume and ascites, increased cardiac output, dP/dt, and ejection fraction, while reversing the marked increase in aldehydes, a marker of oxidative stress, observed in the placebo group. Although candesartan did not alter LV AT₁R protein expression compared to placebo or sham, it reversed the decrease in AT₂R protein observed in the placebo group. Conclusion Our results indicate that in the pacing model of heart failure, chronic AT₁R blockade attenuates hemodynamic deterioration and limits LV remodeling and dysfunction, in part by reversing oxidative stress and AT₂R downregulation.     

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.     

Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells

Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.