Objective: To investigate whether androgens can regulate the expression of eNOS in rat corpus cavernosum through AKT3, PIK3CA, CALM, and CAV1 and influence erectile function. METHODS: Thirty-six 8-week-old male SD rats were randomly divided into groups A (4-week control), B (6-week control), C (4-week castration), D (6-week castration), E (4-week castration + testosterone replacement), and F (6-week castration + testosterone replacement). Both the testis and epididymis were removed from the rats in groups C, D, E and F, and on the second day after surgery, the animals of groups E and F were subcutaneously injected with testosterone propionate at 3 mg per kg of the body weight qd alt while all the others with isodose oil instead. At 4 weeks (for groups A, C and E) and 6 weeks (for groups B, D and F) after treatment, we detected the maximum intracavernous pressure (ICPmax), the mean carotid arterial pressure (MAP) and their ratio (ICPmax/MAP), measured the level of serum testosterone (T), and determined the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: No statistically significant differences were observed in the body weight and MAP among different groups. The serum T level and ICPmax/MAP were remarkably lower in groups C and D than in the other four groups (P<0.01) as well as in groups E and F than in A and B (P<0.05) but exhibited no significant differences either between E and F or between A and B. Immunohistochemistry showed that eNOS and P-eNOS were mainly expressed in the vascular endothelial cell membrane and cavernous vascular lumen, while AKT3, PIK3CA, CALM and CAV1 chiefly in the vascular endothelial cell cytoplasm and membrane, with a few in the smooth muscle cells. Western blot analysis manifested that the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 were markedly lower in groups C and D than in A, B, E and F (P<0.01) as well as in D than in C (P<0.05) but those in groups E and F did not showed any significant difference from those in A and B, nor E from F or A from B. CONCLUSIONS Androgens can improve erectile function by upregulating the expressions of AKT3, PIK3CA, CALM and CAV1 protein molecules and activating eNOS after its phosphorylation, though the exact molecular mechanisms are yet to be further studied.
Animals ; Blood Pressure ; Blotting, Western ; Caveolin 1 ; metabolism ; Class I Phosphatidylinositol 3-Kinases ; metabolism ; Erectile Dysfunction ; Hormone Replacement Therapy ; Male ; Monomeric Clathrin Assembly Proteins ; metabolism ; Myocytes, Smooth Muscle ; Nitric Oxide Synthase Type III ; metabolism ; Orchiectomy ; Penile Erection ; physiology ; Penis ; enzymology ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone Propionate ; administration & dosage

BACKGROUNDThe mechanisms underlying diabetic encephalopathy are largely unknown, and no effective treatments are available. Catalpol has received much attention due to its numerous biological effects, especially in neuroprotective studies. The aim of this study was to investigate the effects of catalpol on cognitive functions in diabetic rats and the underlying mechanisms.

METHODSA rat model of diabetes was established by streptozotocin injection, followed by intraperitoneal infusion of catalpol after 10 weeks. Two weeks later, the Morris water maze was used to test the spatial learning performance. Nissl staining was performed to evaluate the morphological changes in the hippocampus. Expression of protein kinase Cγ (PKCγ) and caveolin-1 (Cav-1) in the hippocampus were assessed by reverse transcription PCR and Western blotting. Activities of anti-oxidative enzymes such as glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) and levels of malonaldehyde (MDA) were measured using commercial kits.

RESULTSSignificant hippocampal neuronal injury was observed in rats with streptozotocin-induced diabetes. Moreover, cognitive dysfunction was associated with markedly increased oxidative stress in the brain. Catalpol treatment significantly attenuated cognitive deficits, neuronal damage, and oxidative stress in the brain of diabetic rats. Biochemical analyses showed that catalpol reversed the down-regulation of PKCγ and Cav-1 expression in the diabetic rats.

CONCLUSIONSSpatial memory in diabetic rats is associated with the expression of PKCγ and Cav-1. Catalpol treatment markedly attenuated oxidative stress, reversed the alteration of PKCγ, Cav-1 and spatial memory deficits.

Animals ; Caveolin 1 ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; physiopathology ; Hippocampus ; drug effects ; metabolism ; Iridoid Glucosides ; therapeutic use ; Male ; Neuroprotective Agents ; therapeutic use ; Oxidative Stress ; drug effects ; Protein Kinase C ; metabolism ; Rats ; Spatial Memory ; drug effects ; physiology

Caveolae are specialized lipid rafts that form flask-shaped invaginations of the plasma membrane. Many researches show that caveolae are involved in cell signaling and transport. Caveolin-1 is the major coat protein essential for the formation of caveolae. Recently, several reports indicated that the other caveolae-associated proteins, Cavins, are required for caveola formation and organization. It's worth noting that Cavin-1 could cooperate with Caveolin-1 to accommodate the structural integrity and function of caveolae. Here, we reviewed that the relationship between Cavins and Caveolins and explore the role of them in regulating caveolae.
Animals ; Caveolae ; physiology ; Caveolin 1 ; metabolism ; physiology ; Caveolins ; metabolism ; physiology ; Humans ; Membrane Proteins ; metabolism ; physiology ; RNA-Binding Proteins ; metabolism ; physiology

It has been proposed that the pro-inflammatory catalytic activity of cyclooxygenase-2 (COX-2) plays a key role in the aging process. However, it remains unclear whether the COX-2 activity is a causal factor for aging and whether COX-2 inhibitors could prevent aging. We here examined the effect of COX-2 inhibitors on aging in the intrinsic skin aging model of hairless mice. We observed that among two selective COX-2 inhibitors and one non-selective COX inhibitor studied, only NS-398 inhibited skin aging, while celecoxib and aspirin accelerated skin aging. In addition, NS-398 reduced the expression of p53 and p16, whereas celecoxib and aspirin enhanced their expression. We also found that the aging-modulating effect of the inhibitors is closely associated with the expression of type I procollagen and caveolin-1. These results suggest that pro-inflammatory catalytic activity of COX-2 is not a causal factor for aging at least in skin and that COX-2 inhibitors might modulate skin aging by regulating the expression of type I procollagen and caveolin-1.
Animals ; Aspirin/administration & dosage ; Catalysis ; Caveolin 1/genetics/metabolism ; Collagen Type I/genetics/metabolism ; *Cyclooxygenase 2/metabolism/physiology ; Cyclooxygenase 2 Inhibitors/*administration & dosage ; Gene Expression Regulation ; Mice ; Nitrobenzenes/*administration & dosage ; Pyrazoles/administration & dosage ; Skin Aging/*drug effects/physiology ; Sulfonamides/*administration & dosage ; Tumor Suppressor Protein p53/genetics/metabolism

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Animals ; Apoptosis ; drug effects ; Caveolin 1 ; genetics ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; physiology ; Filipin ; pharmacology ; MAP Kinase Signaling System ; Macrophages ; cytology ; drug effects ; Mice ; Thapsigargin ; pharmacology ; Transcription Factor CHOP ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

This study examined the radiation-induced ERBB2 nuclear transport in the BT474 breast cancer cell line and the relationship between caveolin-1 and radiation-induced ERBB2 nuclear transport. The BT474 cells were treated with herceptin (200 nmol/L), PP2 (a caveolin-1 inhibitor, 100 nmol/L) and irradiation combined or alone. Confocal microscopy was used to observe the nuclear import of ERBB2 and caveolin-1 after irradiation. Western blotting was employed to detect the expression of ERBB2, caveolin-1 and DNA-PKcs after irradiation, and immunoprecipitation to identify the ERBB2 and caveolin-1 complex before perinuclear ERBB2 localization. Confocal microscopy showed the transport of ERBB2 and caveolin-1 from the cell membrane to the nucleus 15 min after irradiation and the proteins accumulated at the perinuclear region within 45 min. Western blotting revealed that the expression levels of ERBB2, caveolin-1 and DNA-PKcs were increased after irradiation and reached a peak 45 min later. Both herceptin and PP2 treatments were found to decrease ERBB2 expression. An immune complex composed of ERBB2 and caveolin-1 was found in the herceptin group after irradiation. It was concluded that after irradiation, ERBB2 may be transported from the cell membrane to the nucleus and activate DNA-PKcs to trigger DNA double-strand break (DSB) repair; caveolin-1 may participate in this process. Treatments involving the downregulation of caveolin-1 may increase the radiosensitization of breast cancer cells.
Active Transport, Cell Nucleus ; physiology ; Breast Neoplasms ; metabolism ; physiopathology ; Caveolin 1 ; metabolism ; Cell Line, Tumor ; Female ; Humans ; Protein Transport ; physiology ; Radiation ; Receptor, ErbB-2 ; metabolism

Although the function of extracellular Ca(2+)-sensing receptor (CaR) is known, the regulatory mechanism of the CaR function remains to be clarified. The purpose of the present study was to investigate the effect of caveolin-1 (Cav-1) on CaR-induced extracellular Ca(2+) influx by using acute caveolae disruption with Filipin or siRNA targeted to the Cav-1 in human umbilical vein endothelial cells (HUVECs). Intracellular Ca(2+) concentration ([Ca(2+)](i)) was detected by Fura-2/AM loading. The results showed that different concentrations of extracellular Ca(2+) failed to increase [Ca(2+)](i), while the CaR agonist Spermine (2 mmol/L) resulted in an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) (P<0.05). No matter in buffer with or without 2 mmol/L Ca(2+), the [Ca(2+)](i) increase induced by Spermine in HUVECs was abolished after inhibition of CaR by a negative allosteric modulator Calhex231 (1 μmol/L) (P<0.05), conversely, the effect of Spermine on the increase in [Ca(2+)](i) in HUVECs was further augmented after acute caveolae disruption with Filipin (1.5 μg/mL) or transfection with siRNA targeted to the Cav-1 (P<0.05). This indicated that Cav-1 produced an inhibition of CaR-induced extracellular Ca(2+) influx. As to the biological mechanism of Cav-1-induced inhibition, immunofluorescence technique showed that both CaR and Cav-1 were present in HUVECs, and confocal microscopy supported the co-localization of CaR and Cav-1 on the plasma membrane. Functionally, the Cav-1 protein expression was decreased in HUVECs transfected with siRNA targeted to the Cav-1 (P<0.05); simultaneously, the CaR membrane protein expression was decreased (P<0.05), whereas CaR total protein level was unaffected (P>0.05). In conclusion, the present study suggests that CaR and Cav-1 co-localize on the plasma membrane in HUVECs and CaR-induced Ca(2+) influx is down-regulated by binding with Cav-1, and the mechanism involves the effect of Cav-1 on CaR localization on the plasma membrane and attenuating the CaR response to the agonist.
Calcium ; metabolism ; Calcium Channels ; metabolism ; Caveolin 1 ; agonists ; physiology ; Cells, Cultured ; Down-Regulation ; Filipin ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Receptors, Calcium-Sensing ; physiology ; Spermine ; pharmacology

OBJECTIVETo investigate the effects of caveolin-1 on the biologic behavior of pancreatic carcinoma cell line panc1 cells in vitro.

METHODSEukaryotic expression vectors containing human caveolin-1 gene was stably transfected into panc1 cells with Lipofectamine2000. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western plotting. The cell growth activity was examined by MTT assay. Anchorage-independent growth was detected by colony formation assay in soft agar. Flow cytometry was used to analyze the cell cycle and apoptosis. Cell invasion assay was used for evaluating cell invasion capacity. The relative phosphorylation level of EGFR, c-Raf, Mek, Erk, p38 and SAPK/JNK were detected by Western blotting.

RESULTSThree transfected cell clones overexpressing caveolin-1 were obtained. Comparing with the panc1 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of flow cytometry showed that over-expression of caveolin-1 resulted in the cell cycle arrest at G(0)/G(1) phase and increased the apoptotic cell fraction. Cell invasion assay showed that overexpression of caveolin-1 significantly inhibited the panc1 cell invasion. Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR, c-Raf, Mek and Erk while did not affect the activity of p38 and SAPK/JNK.

CONCLUSIONOver-expression of caveolin-1 inhibits the growth and invasion of pancreatic carcinoma cells in vitro. These phenotypes may be correlated with the inhibition of EGFR-c-Raf-Mek-Erk signaling pathway.

Apoptosis ; Caveolin 1 ; genetics ; metabolism ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Neoplasm Invasiveness ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; Proto-Oncogene Proteins c-raf ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; Transfection

Caveolin 1 ; genetics ; metabolism ; Cells, Cultured ; Homocysteine ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro.

METHODSEukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy.

RESULTSThe expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2.

CONCLUSIONOverexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.

Apoptosis ; Blotting, Western ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Caveolin 1 ; genetics ; metabolism ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Flow Cytometry ; Genetic Vectors ; chemistry ; genetics ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Lipids ; chemistry ; Microscopy, Confocal ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; physiology ; Transfection ; methods