scholarly journals In Vivo Ablation of the Conserved GATA-Binding Motif in the Amh Promoter Impairs Amh Expression in the Male Mouse

Endocrinology ◽  
2019 ◽  
Vol 160 (4) ◽  
pp. 817-826 ◽  
Author(s):  
Marie France Bouchard ◽  
Francis Bergeron ◽  
Jasmine Grenier Delaney ◽  
Louis-Mathieu Harvey ◽  
Robert S Viger
Keyword(s):  
Developmental Time ◽  
Gonadal Development ◽  
Conclusive Evidence ◽  
Binding Motif ◽  
Male Embryo ◽  
Steroidogenic Factor 1 ◽  
Protein Levels ◽  
Positive Modulator ◽  
And Function

Abstract GATA4 is an essential transcriptional regulator required for gonadal development, differentiation, and function. In the developing testis, proposed GATA4-regulated genes include steroidogenic factor 1 (Nr5a1), SRY-related HMG box 9 (Sox9), and anti-Müllerian hormone (Amh). Although some of these genes have been validated as genuine GATA4 targets, it remains unclear whether GATA4 is a direct regulator of endogenous Amh transcription. We used a CRISPR/Cas9-based approach to specifically inactivate or delete the sole GATA-binding motif of the proximal mouse Amh promoter. AMH mRNA and protein levels were assessed at developmental time points corresponding to elevated AMH levels: fetal and neonate testes in males and adult ovaries in females. In males, loss of GATA binding to the Amh promoter significantly reduced Amh expression. Although the loss of GATA binding did not block the initiation of Amh transcription, AMH mRNA and protein levels failed to upregulate in the developing fetal and neonate testis. Interestingly, adult male mice presented no anatomical anomalies and had no evidence of retained Müllerian duct structures, suggesting that AMH levels, although markedly reduced, were sufficient to masculinize the male embryo. In contrast to males, GATA binding to the Amh promoter was dispensable for Amh expression in the adult ovary. These results provide conclusive evidence that in males, GATA4 is a positive modulator of Amh expression that works in concert with other key transcription factors to ensure that the Amh gene is sufficiently expressed in a correct spatiotemporal manner during fetal and prepubertal testis development.

scholarly journals Interaction Between Dax-1 and Steroidogenic Factor-1 in Vivo: Increased Adrenal Responsiveness to ACTH in the Absence of Dax-1

Endocrinology ◽  
2002 ◽  
Vol 143 (2) ◽  
pp. 665-673 ◽  
Author(s):  
Poda Suresh Babu ◽  
David L. Bavers ◽  
Felix Beuschlein ◽  
Sonalee Shah ◽  
Baxter Jeffs ◽  
...  
Keyword(s):  
In Vitro Assays ◽  
Acth Stimulation ◽  
Steroidogenic Factor 1 ◽  
Protein Levels ◽  
Cellular Hypertrophy ◽  
Acth Receptor ◽  
Adrenal Hypoplasia ◽  
And Function

Abstract Two nuclear receptors, dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1 (Dax-1) and steroidogenic factor-1 (SF-1), are required for adrenal development and function. In vitro assays suggest that Dax-1 represses SF-1 mediated transcription. In this study, we generated SF-1+/−: Dax-1−/Y mice to examine the role of Dax-1 in SF-1-dependent steroidogenesis in vivo. While the SF-1 expression was impaired in SF-1+/− mice, there was no change in Dax-1 expression in SF-1+/− mice and no change in SF-1 expression in Dax-1−/Y mice. SF-1+/− mice had small adrenal glands with adrenal hypoplasia and cellular hypertrophy. The loss of Dax-1 in SF-1+/−: Dax-1−/Y mice reversed the decreased adrenal weight and histological abnormalities observed in SF-1+/− mice. SF-1+/− mice had elevated ACTH and the lowest corticosterone following restraint stress. In contrast, Dax-1−/Y mice had elevated corticosterone and decreased ACTH. Adrenal responsiveness (ACTH/corticosterone) was highest in Dax-1−/Y mice, intermediate in WT and SF-1+/−: Dax-1−/Y mice, and lowest in SF-1+/− mice. In accordance with these findings, ACTH stimulation testing resulted in the highest levels of corticosterone in the Dax-1−/Y mice. Protein levels of P450c21 and the ACTH receptor were increased in Dax-1−/Y mice and intermediate in SF-1+/−: Dax-1−/Y mice following chronic food deprivation. These results are consistent with a model in which Dax-1 functions to inhibit SF-1-mediated steroidogenesis in vivo.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

2021 ◽  
Author(s):  
Bin Qiu ◽  
Zhaohui Zhong ◽  
Shawn Righter ◽  
Yuxue Xu ◽  
Jun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Disease Onset ◽  
Fold Increase ◽  
Knock Out ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

scholarly journals IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2409-2414 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Steven A. Rosenberg
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
High Dose ◽  
Protein Levels ◽  
And Function ◽  
The Impact

Abstract Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

scholarly journals Mesenchymal Stem Cells with Enhanced Bcl-2 Expression Promote Liver Recovery in a Rat Model of Hepatic Cirrhosis

2016 ◽  
Vol 40 (5) ◽  
pp. 1117-1128 ◽  
Author(s):  
Shizhu Jin ◽  
Hulun Li ◽  
Mingzi Han ◽  
Mengting Ruan ◽  
Zishuai Liu ◽  
...  
Keyword(s):  
Liver Function ◽  
Hepatic Cirrhosis ◽  
Post Transplantation ◽  
Experimental Cirrhosis ◽  
Adeno Associated Virus ◽  
Protein Levels ◽  
Histological Sign ◽  
And Function

Background/Aims: Mesenchymal stem cell (MSC) transplantation has emerged as an option for the treatment of chronic hepatic cirrhosis, while its therapeutic efficacy could be improved. The bcl-2 gene is anti-apoptotic and can help cell survival and proliferation. Therefore, we explored whether transplanted MSCs with enhanced bcl-2 expression may be beneficial in the treatment of experimental cirrhosis in rats. Methods: MSCs were isolated from rat bone marrow, expanded in vitro and transfected with adeno-associated virus (AAV) engineered the bcl-2 gene (AAV-bcl-2). Rats with cirrhosis induced by carbon tetrachloride (CCl4) were treated with AAV-bcl-2 infected BMSCs-AAV-bcl-2, with the cells traced in vivo post transplantation. Liver pathology and function were evaluated 7, 14, 21, and 28 days post transplantation, respectively. Results: On day 7 post transplantation, the infused AAV-bcl-2 had integrated into the hepatocyte-like cells (HLCs) that expressed albumin (ALB), Cytokeratin 18 (CK18), and hepatocytes nuclear factor 4a (HNF4a). On day 28 post transplantation, rats in the cirrhosis + BMSCs-AAV-bcl-2 group showed the most dense HLCs, highest mRNA and protein levels of ALB, CK18, and HNF4a, compared to the other groups. Their liver function recovered most rapidly in 4 week observation, while histological sign of cirrhosis remained at the end of this period. Conclusion: BMSCs over expressing bcl-2 gene showed better survival, and enhanced the differentiation into hepatocytes-like cells, and appeared to promote the recovery of liver function in rats with experimental cirrhosis.

scholarly journals Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

2011 ◽  
Vol 22 (23) ◽  
pp. 4503-4512 ◽  
Author(s):  
Zhifang Chai ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junction ◽  
Hela Cells ◽  
Pdz Domain ◽  
Cell Communication ◽  
Normal Function ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Interacting Protein ◽  
And Function

The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

scholarly journals The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

2018 ◽  
Author(s):  
Lewie Zeng ◽  
Rachid El Bejjani ◽  
Marc Hammarlund
Keyword(s):  
Motor Neurons ◽  
Neuronal Development ◽  
Axon Regeneration ◽  
Neuronal Response ◽  
Small Gtpase ◽  
C Elegans ◽  
Protein Levels ◽  
Mature Neurons ◽  
And Function

AbstractMembers of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.

scholarly journals Drosophila MIC60/mitofilin conducts dual roles in mitochondrial motility and crista structure

2017 ◽  
Vol 28 (24) ◽  
pp. 3471-3479 ◽  
Author(s):  
Pei-I Tsai ◽  
Amanda M. Papakyrikos ◽  
Chung-Han Hsieh ◽  
Xinnan Wang
Keyword(s):  
Neuromuscular Junctions ◽  
Cellular Level ◽  
Dual Roles ◽  
Protein Levels ◽  
Mitochondrial Homeostasis ◽  
Synaptic Structure ◽  
Microtubule Motors ◽  
Mitochondrial Motility ◽  
And Function

MIC60/mitofilin constitutes a hetero-oligomeric complex on the inner mitochondrial membranes to maintain crista structure. However, little is known about its physiological functions. Here, by characterizing Drosophila MIC60 mutants, we define its roles in vivo. We discover that MIC60 performs dual functions to maintain mitochondrial homeostasis. In addition to its canonical role in crista membrane structure, MIC60 regulates mitochondrial motility, likely by influencing protein levels of the outer mitochondrial membrane protein Miro that anchors mitochondria to the microtubule motors. Loss of MIC60 causes loss of Miro and mitochondrial arrest. At a cellular level, loss of MIC60 disrupts synaptic structure and function at the neuromuscular junctions. The dual roles of MIC60 in both mitochondrial crista structure and motility position it as a crucial player for cellular integrity and survival.

scholarly journals Functional analysis of zebrafish microfibril-associated glycoprotein-1 (Magp1) in vivo reveals roles for microfibrils in vascular development and function

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4364-4374 ◽  
Author(s):  
Eleanor Chen ◽  
Jon D. Larson ◽  
Stephen C. Ekker
Keyword(s):  
Vascular Development ◽  
Cardiovascular Tissue ◽  
Protein Levels ◽  
Wide Range ◽  
Vessel Structure ◽  
Fibrillin 1 ◽  
Critical Requirement ◽  
And Function ◽  
Microfibril Formation

AbstractMutations in fibrillin-1 (FBN1) result in Marfan syndrome, demonstrating a critical requirement for microfibrils in vessel structure and function. However, the identity and function of many microfibril-associated molecules essential for vascular development and function have yet to be characterized. In our morpholino-based screen for members of the secretome required for vascular development, we identified a key player in microfibril formation in zebrafish embryogenesis. Microfibril-associated glycoprotein-1 (MAGP1) is a conserved protein found in mammalian and zebrafish microfibrils. Expression of magp1 mRNA is detected in microfibril-producing cells. Analysis of a functional Magp1-mRFP fusion protein reveals localization along the midline and in the vasculature during embryogenesis. Underexpression and overexpression analyses demonstrate that specific Magp1 protein levels are critical for vascular development. Integrin function is compromised in magp1 morphant embryos, suggesting that reduced integrin–matrix interaction is the main mechanism for the vascular defects in magp1 morphants. We further show that Magp1 and fibrillin-1 interact in vivo. This study implicates MAGP1 as a key player in microfibril formation and integrity during development. The essential role for MAGP1 in vascular morphogenesis and function also supports a wide range of clinical applications, including therapeutic targets in vascular disease and cardiovascular tissue engineering.

scholarly journals Forskolin, 8-Br-3′,5′-Cyclic Adenosine 5′-Monophosphate, and Catalytic Protein Kinase A Expression in the Nucleus Increase Radioiodide Uptake and Sodium/Iodide Symporter Protein Levels in RET/PTC1-Expressing Cells

2004 ◽  
Vol 89 (12) ◽  
pp. 6168-6172 ◽  
Author(s):  
Anjli Venkateswaran ◽  
Derek K. Marsee ◽  
Steven H. Green ◽  
Sissy M. Jhiang
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Sodium Iodide ◽  
Sodium Iodide Symporter ◽  
Thyroid Cells ◽  
Protein Levels ◽  
And Function ◽  
Kinase A

Abstract RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways. We showed that both forskolin and 8-Br-cAMP increase radioiodide uptake and NIS protein in RET/PTC1-expressing cells to the same extent as the parental PC Cl 3 cells. We found that RET/PTC1 decreases nuclear localization of catalytic PKA, and forskolin treatment was able to counteract this RET/PTC1 effect. Furthermore, transient expression of catalytic PKA in the nucleus increased radioiodide uptake and NIS protein in RET/PTC1-expressing cells. Taken together, these studies suggest that RET/PTC1 down-regulates NIS expression by interrupting TSH/cAMP signaling, and this RET/PTC1 effect can be reversed by activating cAMP-PKA pathways.

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