119. Over-expression of activin βC in vivo reveals a role in male fertility

2005 ◽  
Vol 17 (9) ◽  
pp. 74
Author(s):  
E. Gold ◽  
M. O'Bryan ◽  
S. Meachem ◽  
H. McDougall ◽  
C. Butler ◽  
...  
Keyword(s):  
Liver Cell ◽  
Male Fertility ◽  
Protein Extraction ◽  
Activin A ◽  
Cmv Promoter ◽  
Over Expression ◽  
Age Related

Introduction: Activin βC subunit antagonises the formation and bioactivity of activin A via intracellular heterodimerisation and decreases activation of the activin signalling pathway (Mellor et al. 2003). Therefore the activin βC subunit heterodimers provide a new mechanism of regulating activin levels. Vedja and colleagues over-expressed the activin βC subunit in malignant liver cell lines, which subsequently displayed inhibition of cell proliferation and induction of apoptosis (Vedja et al. 2003). Conversely, Wada et al. demonstrated that treatment with hr-activin C stimulates growth of a liver cell line (Wada et al. 2004). These recent (and contradictory) reports about the in vitro activity of activin βC have prompted us to examine the in vivo role of activin βC by creating a transgenic mouse over-expressing the βC activin subunit. Methods: The full-length human cDNA under the control of a CMV promoter was incorporated into the genome of three founder C57/B6 mice. Genotyping was performed by both Southern and PCR. Mice were monitored weekly and culled at 14–16 weeks (adult). Blood was collected by cardiac puncture, organs were weighed and a portion fixed in Bouin’s or frozen for subsequent RNA and protein extraction. Daily sperm production (DSP) was determined by standard methods. Sertoli and germ cell number will be determined using the optical disector (sic) stereological technique in Bouin’s fixed resin sections. Proliferation and apoptosis will be examined using PCNA and TUNEL respectively. Activin A was assessed by ELISA, while FSH, LH, follistatin and total inhibin were determined by RIA. Results and conclusions: Over-expression of activin-βC resulted in decreased circulating activin A (P < 0.005 TG1, P < 0.05 TG2 and P = 0.08 TG3), a progressive age-related decrease in litter sizes (9.3 WT v. 6.3 TG1, 5.8 TG2 and 4.5 TG3; P < 0.005 v. WT) and testicular DSP (P < 0.05). These data support the hypothesis that βC is a novel in vivo regulator and is the first indication of a role for activin-βC in male fertility. This novel mouse model will significantly advance our understanding of the in vivo role of activin-βC. (1)Mellor et al. (2003). Endocrinology 144, 4410–4419.(2)Vejda et al. (2003). Carcinogenesis 24, 1801–1809.(3)Wada et al. (2004). Am. J. Physiol. Endocrinol. Metab. 287, E247–E254.

scholarly journals Complement C5 is not critical for the formation of sub-RPE deposits in Efemp1 mutant mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Donita L. Garland ◽  
Eric A. Pierce ◽  
Rosario Fernandez-Godino
Keyword(s):  
Macular Degeneration ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Deposit Formation ◽  
Genetic Ablation ◽  
Age Related ◽  
Complement Dysregulation

AbstractThe complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

scholarly journals A Critical Role of Activin A in Maturation of Mouse Peritoneal Macrophages in vitro and in vivo

2009 ◽  
Vol 6 (5) ◽  
pp. 387-392 ◽  
Author(s):  
Yinan Wang ◽  
Xueling Cui ◽  
Guixiang Tai ◽  
Jingyan Ge ◽  
Nan Li ◽  
...  
Keyword(s):  
Critical Role ◽  
Peritoneal Macrophages ◽  
Activin A ◽  
Mouse Peritoneal Macrophages

scholarly journals STIL-a novel link in Shh and Wnt signaling, endowing oncogenic and stem like attributes to colorectal cancer

2020 ◽  
Author(s):  
Tapas Pradhan ◽  
Vikas Kumar ◽  
H Evangeline Surya ◽  
R Krishna ◽  
Samu John ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Importance ◽  
Disease Free Survival ◽  
Drug Resistant ◽  
Free Survival ◽  
Over Expression ◽  
Disease Free

AbstractDiscovery of potent gene regulating tumorigenesis and drug resistance is of high clinical importance. STIL is an oncogene, however its molecular insights and role in colorectal oncogenesis are unknown. In this study we have explored role of STIL in tumorigenesis and studied its molecular targets in colorectal cancer (CRC). STIL silencing reduced proliferation and tumor growth in CRC. Further, STIL was found to regulate stemness markers CD133 & CD44 and drug resistant markers Thymidylate synthase, ABCB1 & ABCG2 both in in-vitro and in-vivo CRC models. In addition, over expression of STIL mRNA was found to be associated with reduced disease free survival in CRC cases. To our surprise we observed an Shh independent regulation of stemness and drug resistant genes mediated by STIL. Interestingly, we found an Shh independent regulation of β-catenin mediated by STIL via p-AKT, which partially answers Shh independent regulatory mechanism of CSC markers by STIL. Our study suggest an instrumental role of STIL in molecular manifestation of CRC and progression.

scholarly journals Notch Signaling Regulates MMP-13 Expression via Runx2 in Chondrocytes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Di Xiao ◽  
Ruiye Bi ◽  
Xianwen Liu ◽  
Jie Mei ◽  
Nan Jiang ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Chondrocyte Proliferation ◽  
Runx2 Expression ◽  
Over Expression ◽  
Proliferation And Differentiation ◽  
Important Regulator ◽  
Signaling Activation

Abstract Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment. In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.

MiR-214 Attenuates the Osteogenic Effects of Mechanical Loading on Osteoblasts

2019 ◽  
Vol 40 (14) ◽  
pp. 931-940
Author(s):  
Yu Yuan ◽  
Jianmin Guo ◽  
Lingli Zhang ◽  
Xiaoyang Tong ◽  
Shihua Zhang ◽  
...  
Keyword(s):  
Mechanical Loading ◽  
Mirna Expression ◽  
Mechanical Strain ◽  
Mechanisms Of Action ◽  
Matrix Mineralization ◽  
Over Expression ◽  
Osteogenic Factors

AbstractExercise is an effective way to prevent osteoporosis, but its mechanism remains unclear. MicroRNAs (miRNAs) play an essential role in bone metabolism. Recently, mechanical loading was reported to induce changes in miRNA expression in osteoblasts. However, the role of miRNAs in bone under exercise and its underlining mechanisms of action still remain unknown. MiR-214 was reported to regulate the process of osteogenesis and is considered a biomarker of osteoporosis. In this study, we aimed to investigate whether exercise could induce changes in miRNA expression in bone and to study the effects of miR-214 on mechanical loading-induced osteogenesis in osteoblasts. The results showed that miR-214 was down-regulated in both tibia from C57BL/6 mice after exercise in vivo and in osteoblasts after mechanical strain in vitro. Mechanical strain could enhance the ALP activity, promote matrix mineralization, up-regulate the expression of osteogenic factors such as ATF4, Osterix, ALP and β-catenin, and down-regulate RANKL and RANK expression. Over-expression of miR-214 not only inhibited the expression of these osteogenic factors but also attenuated mechanical strain-enhanced osteogenesis in osteoblasts. Collectively, our results indicated that miR-214 could attenuate the osteogenic effects of mechanical loading on osteoblasts, suggesting that inhibition of miR-214 may be one of the ways in which exercise prevents osteoporosis.

scholarly journals FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity

2021 ◽  
Vol 9 ◽  
Author(s):  
Marianna Kapetanou ◽  
Tobias Nespital ◽  
Luke S. Tain ◽  
Andre Pahl ◽  
Linda Partridge ◽  
...  
Keyword(s):  
Proteasome Activity ◽  
20S Proteasome ◽  
Direct Role ◽  
Forkhead Box ◽  
Age Related ◽  
Proteasome Subunits ◽  
Rate Limiting

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

scholarly journals Complement C5 is Not Critical for the Formation of Sub-RPE Deposits ­in Efemp1 Mutant Mice

2021 ◽  
Author(s):  
Donita Garland ◽  
Eric Pierce ◽  
Rosario Fernandez-Godino
Keyword(s):  
Macular Degeneration ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Deposit Formation ◽  
Genetic Ablation ◽  
Age Related ◽  
Complement Dysregulation

Abstract The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

scholarly journals Farnesoid X Receptor Protects Liver Cells from Apoptosis Induced by Serum Deprivation in Vitro and Fasting in Vivo

2008 ◽  
Vol 22 (7) ◽  
pp. 1622-1632 ◽  
Author(s):  
Yan-Dong Wang ◽  
Fan Yang ◽  
Wei-Dong Chen ◽  
Xiongfei Huang ◽  
Lily Lai ◽  
...  
Keyword(s):  
Liver Cell ◽  
Mapk Pathway ◽  
Specific Inhibitor ◽  
Serum Deprivation ◽  
Farnesoid X Receptor ◽  
Dependent Manner ◽  
Induced Apoptosis

Abstract The farnesoid X receptor (FXR) is a key metabolic regulator in the liver by maintaining the homeostasis of liver metabolites. Recent findings suggest that FXR may have a much broader function in liver physiology and pathology. In the present work, we identify a novel role of FXR in protecting liver cell from apoptosis induced by nutritional withdrawal including serum deprivation in vitro or starvation in vivo. Two FXR ligands, chenodeoxycholic acid (CDCA) and GW4064, rescued HepG2 cells from serum deprivation-induced apoptosis in a dose-dependent manner. This effect of FXR on apoptotic suppression was compromised when FXR was knocked down by short interfering RNA. Similarly, the effects of both CDCA and GW4064 were abolished after inhibition of the MAPK pathway by a specific inhibitor of MAPK kinase 1/2. Immunoblotting results indicated that FXR activation by CDCA and GW4064 induced ERK1/2 phosphorylation, which was attenuated by serum deprivation. In vivo, FXR−/− mice exhibited an exacerbated liver apoptosis and lower levels of phosphorylated-ERK1/2 compared to wild-type mice after starvation. In conclusion, our results suggest a novel role of FXR in modulating liver cell apoptosis.

scholarly journals Role of SMAD2 and SMAD3 on Adipose Tissue Development and Function

2021 ◽  
Author(s):  
◽  
Roshan Kumari ◽  
Keyword(s):  
Body Weight ◽  
Adipose Tissue ◽  
Fat Mass ◽  
Energy Homeostasis ◽  
Activin A ◽  
Male Mice ◽  
Proliferation And Differentiation

Introduction: Obesity and its associated metabolic syndrome are major medical problems worldwide including United States. Adipose tissue is the primary site of energy storage, playing important roles in health. Adipose tissue also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety, inflammation, and total energy homeostasis. Activin A and activin B play important roles in maintaining body composition and energy homeostasis. This dissertation highlights the role of activin/SMADs signaling in adipose tissue development, function, and maintenance. SMAD2/3 proteins are downstream mediators of transforming growth factor-β (TGFβ) family signaling, including activins, which regulate critical preadipocyte and mature adipocyte functions. Previous studies have demonstrated that Smad2 global knockout mice exhibit embryonic lethality, whereas global loss of Smad3 protects mice against diet-induced obesity and the direct contributions of Smad2 and Smad3 in adipose tissues individually or in combination and the responses of these tissues to activin signaling are unknown. Additionally, our lab demonstrated that the combined loss of activin A and activin B have reduced adiposity in mice and appearance of brown-like cells in visceral white adipose tissue. However, the cell-autonomous role of activins on cell proliferation and differentiation remained unknown in vitro. My hypothesis was that activin signaling regulate adipocyte differentiation and functions via SMAD2/3-mediated mechanism(s) and that the individual or combined adipose-specific deletion of SMAD2/SMAD3 would result in reduced adiposity similar to activin deficient mice. Objective: Here, we sought to determine the primary effects of adipocyte-selective reduction of Smad2 or Smad3 individually and in combination, on diet-induced adiposity and to establish whether preadipocytes isolated from subcutaneous and visceral white adipose tissues differ in their differentiation capacity. We also assessed the role of activins on cell proliferation and differentiation using an in vitro model. Research Design: To assess the adipose-selective requirements of Smad2, Smad3 and Smad2/3, we generated three lines of adipose-selective conditional knockout (cKO) mice including Smad2cKO, Smad3cKO, and Smad2/3 double cKO mice using Smad2 and/or Smad3 “floxed” mice intercrossed with Adiponectin-Cre mice. Additionally, we isolated preadipocytes and examined adipogenic activity of visceral and subcutaneous preadipocyte and the effects of activin on preadipocyte proliferation and differentiation in vitro. Furthermore, we used mouse embryonic fibroblasts (MEFs) from wild type mice and activin double knockout mice to study the cell autonomous role of activin on differentiation and cell fate. Results: Our results demonstrated that subcutaneous preadipocytes differentiate uniformly and almost all wildtype subcutaneous preadipocytes differentiated into mature adipocytes. In contrast, visceral preadipocytes differentiated poorly. Exogenous activin A promoted proliferation and suppressed differentiation of subcutaneous preadipocytes more robustly given that visceral adipocytes differentiate poorly at baseline. Additionally, global knockout of activin A and B promoted differentiation and browning in differentiated MEFs in vitro consistent with in vivo studies. Furthermore, we showed that Smad2cKO mice did not exhibit significant effects on weight gain, irrespective of diet, whereas Smad3cKO male mice displayed a trend of reduced body weight on high fat diet. On both (LFD and HFD) diets, Smad3cKO male mice displayed an adipose depot-selective phenotype, with significant reduction in subcutaneous fat mass but not visceral fat mass. Smad2/3cKO male mice did not show any difference in body weight or fat mass compared to control mice. Female mice with adipose-selective combined deletion of Smad2/3, displayed reduced body weight and reduction of fat mass in both visceral and subcutaneous depot with higher metabolic rate on HFD compared to control littermates. Conclusions: Our study demonstrated that Smad3 is an important contributor to the development and/or maintenance of subcutaneous white adipose tissue in a sex-selective fashion. Combined reduction of Smad2/3 protects female mice from diet induced obesity and is important for visceral and subcutaneous depots in a sex-selective fashion. These findings have implications for understanding SMAD-mediated, depot selective regulation of adipocyte growth and differentiation. Activin treatment promoted proliferation of preadipocytes, while activin deficiency promoted differentiation and altered the phenotypic characteristics of White adipocytes to brown-like cells in vitro consistent with in vivo.

scholarly journals A novel knockout mouse for the small EDRK-rich factor 2 (Serf2) showing developmental and other deficits

2021 ◽  
Vol 32 (2) ◽  
pp. 94-103
Author(s):  
Karen Cleverley ◽  
Weaverly Colleen Lee ◽  
Paige Mumford ◽  
Toby Collins ◽  
Matthew Rickman ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Startle Response ◽  
Protein Misfolding ◽  
In Vivo Models ◽  
Mixed Genetic Background ◽  
Age Related ◽  
Male Specific

AbstractThe small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2+/− mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2−/− null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism.

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