mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo

The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier “leaky.” Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.

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Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

International Journal of Molecular Sciences ◽

10.3390/ijms22157918 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7918

Author(s):

Jisun Hwang ◽

Bohee Jang ◽

Ayoung Kim ◽

Yejin Lee ◽

Joonha Lee ◽

...

Keyword(s):

Transmembrane Domain ◽

Growth Factor Receptor ◽

Cytoplasmic Domain ◽

Nih3t3 Cells ◽

C Elegans ◽

Downstream Signaling ◽

Downstream Effectors ◽

Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

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Lineage-specific requirements of β-catenin in neural crest development

The Journal of Cell Biology ◽

10.1083/jcb.200209039 ◽

2002 ◽

Vol 159 (5) ◽

pp. 867-880 ◽

Cited By ~ 207

Author(s):

Lisette Hari ◽

Véronique Brault ◽

Maurice Kléber ◽

Hye-Youn Lee ◽

Fabian Ille ◽

...

Keyword(s):

Transcription Factor ◽

Neural Crest ◽

Neural Crest Cells ◽

Neural Crest Stem Cells ◽

Downstream Signaling ◽

Neural Crest Development ◽

Sensory Neurogenesis

β-Catenin plays a pivotal role in cadherin-mediated cell adhesion. Moreover, it is a downstream signaling component of Wnt that controls multiple developmental processes such as cell proliferation, apoptosis, and fate decisions. To study the role of β-catenin in neural crest development, we used the Cre/loxP system to ablate β-catenin specifically in neural crest stem cells. Although several neural crest–derived structures develop normally, mutant animals lack melanocytes and dorsal root ganglia (DRG). In vivo and in vitro analyses revealed that mutant neural crest cells emigrate but fail to generate an early wave of sensory neurogenesis that is normally marked by the transcription factor neurogenin (ngn) 2. This indicates a role of β-catenin in premigratory or early migratory neural crest and points to heterogeneity of neural crest cells at the earliest stages of crest development. In addition, migratory neural crest cells lateral to the neural tube do not aggregate to form DRG and are unable to produce a later wave of sensory neurogenesis usually marked by the transcription factor ngn1. We propose that the requirement of β-catenin for the specification of melanocytes and sensory neuronal lineages reflects roles of β-catenin both in Wnt signaling and in mediating cell–cell interactions.

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The Mammalian Target of Rapamycin Complex 1 Regulates Leptin Biosynthesis in Adipocytes at the Level of Translation: The Role of the 5′-Untranslated Region in the Expression of Leptin Messenger Ribonucleic Acid

Molecular Endocrinology ◽

10.1210/me.2008-0148 ◽

2008 ◽

Vol 22 (10) ◽

pp. 2260-2267 ◽

Cited By ~ 15

Author(s):

Partha Chakrabarti ◽

Takatoshi Anno ◽

Brendan D. Manning ◽

Zhijun Luo ◽

Konstantin V. Kandror

Keyword(s):

Untranslated Region ◽

Molecular Mechanisms ◽

Mammalian Target Of Rapamycin ◽

Dominant Negative ◽

Target Of Rapamycin ◽

Messenger Ribonucleic Acid ◽

Leptin Expression ◽

Adipose Cells

Abstract Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5′-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5′-UTR. We found that the presence of leptin 5′-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5′-terminal oligopyrimidine tract or the 5′-UTR.

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MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma

Blood ◽

10.1182/blood-2009-01-198408 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6669-6680 ◽

Cited By ~ 224

Author(s):

Aldo M. Roccaro ◽

Antonio Sacco ◽

Brian Thompson ◽

Xavier Leleu ◽

Abdel Kareem Azab ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Expression Profiling ◽

Map Kinases ◽

Qrt Pcr ◽

Ribosomal Protein S6 ◽

Genomic Studies

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

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Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo

Endocrinology ◽

10.1210/en.2009-1278 ◽

2010 ◽

Vol 151 (6) ◽

pp. 2911-2922 ◽

Cited By ~ 32

Author(s):

Mark J. McCabe ◽

Gerard A. Tarulli ◽

Sarah J. Meachem ◽

David M. Robertson ◽

Peter M. Smooker ◽

...

Keyword(s):

Tight Junctions ◽

Sertoli Cell ◽

Gnrh Antagonist ◽

Hormone Replacement ◽

Recombinant Fsh ◽

Short Term ◽

Adult Rat ◽

Serum Androgens

Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG ± FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

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Decreased TNF Levels and Improved Retinal Ganglion Cell Survival in MMP-2 Null Mice Suggest a Role for MMP-2 as TNF Sheddase

Mediators of Inflammation ◽

10.1155/2015/108617 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 9

Author(s):

Lies De Groef ◽

Manuel Salinas-Navarro ◽

Griet Van Imschoot ◽

Claude Libert ◽

Roosmarijn E. Vandenbroucke ◽

...

Keyword(s):

Ganglion Cell ◽

Retinal Ganglion Cell ◽

Retinal Ganglion ◽

Spatiotemporal Expression ◽

Retinal Ganglion Cell Death ◽

The Central Nervous System ◽

Retinal Ganglion Cell Survival ◽

Ganglion Cell Death

Matrix metalloproteinases (MMPs) have been designated as both friend and foe in the central nervous system (CNS): while being involved in many neurodegenerative and neuroinflammatory diseases, their actions appear to be indispensable to a healthy CNS. Pathological conditions in the CNS are therefore often related to imbalanced MMP activities and disturbances of the complex MMP-dependent protease network. Likewise, in the retina, various studies in animal models and human patients suggested MMPs to be involved in glaucoma. In this study, we sought to determine the spatiotemporal expression profile of MMP-2 in the excitotoxic retina and to unravel its role during glaucoma pathogenesis. We reveal that intravitreal NMDA injection induces MMP-2 expression to be upregulated in the Müller glia. Moreover, MMP-2 null mice display attenuated retinal ganglion cell death upon excitotoxic insult to the retina, which is accompanied by normal glial reactivity, yet reduced TNF levels. Hence, we propose a novelin vivofunction for MMP-2, as an activating sheddase of tumor necrosis factor (TNF). Given the pivotal role of TNF as a proinflammatory cytokine and neurodegeneration-exacerbating mediator, these findings generate important novel insights into the pathological processes contributing to glaucomatous neurodegeneration and into the interplay of neuroinflammation and neurodegeneration in the CNS.

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rpS6 Regulates Blood-Testis Barrier Dynamics Through Arp3-Mediated Actin Microfilament Organization in Rat Sertoli Cells. An In Vitro Study

Endocrinology ◽

10.1210/en.2014-1791 ◽

2015 ◽

Vol 156 (5) ◽

pp. 1900-1913 ◽

Cited By ~ 38

Author(s):

Ka-Wai Mok ◽

Haiqi Chen ◽

Will M. Lee ◽

C. Yan Cheng

Keyword(s):

Sertoli Cells ◽

Protein Complexes ◽

Mammalian Target Of Rapamycin ◽

Attachment Site ◽

In Vitro Study ◽

Site Directed Mutagenesis ◽

Actin Microfilaments ◽

Blood Testis Barrier

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a “bundled” to an “unbundled/branched” configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

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Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2013-204599 ◽

2014 ◽

Vol 74 (7) ◽

pp. 1432-1440 ◽

Cited By ~ 167

Author(s):

Yue Zhang ◽

Faezeh Vasheghani ◽

Ying-hua Li ◽

Meryem Blati ◽

Kayla Simeone ◽

...

Keyword(s):

Articular Cartilage ◽

Protein Kinase ◽

Mammalian Target Of Rapamycin ◽

Adenosine Monophosphate ◽

Cartilage Degradation ◽

Chondrocyte Apoptosis ◽

A Cell ◽

Mtor Signalling

ObjectivesMammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo.MethodsInducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51–like kinase 1 (ULK1) siRNA to determine mTOR signalling.ResultsmTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage.ConclusionsThis study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

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The Role of AMPK/mTOR Signaling Pathway in Anticancer Activity of Metformin

Physiological Research ◽

10.33549/physiolres.934618 ◽

2021 ◽

pp. 501-508

Author(s):

Nikola Chomanicova ◽

Andrea Gazova ◽

Adriana Adamickova ◽

Simona Valaskova ◽

Jan Kyselovic

Keyword(s):

Anticancer Activity ◽

Mammalian Target Of Rapamycin ◽

Adenosine Monophosphate ◽

Mtor Pathway ◽

Diabetic Patients ◽

Beneficial Effects ◽

Treatment Of Diabetes

Metformin (MTF) is a widely used drug for the treatment of diabetes mellitus type 2 (DM2) and frequently used as an adjuvant therapy for polycystic ovarian syndrome, metabolic syndrome, and in some cases also tuberculosis. Its protective effect on the cardiovascular system has also been described. Recently, MTF was subjected to various analyzes and studies that showed its beneficial effects in cancer treatment such as reducing cancer cell proliferation, reducing tumor growth, inducing apoptosis, reducing cancer risk in diabetic patients, or reducing likelihood of relapse. One of the MTF’s mechanisms of action is the activation of adenosine-monophosphate-activated protein kinase (AMPK). Several studies have shown that AMPK/mammalian target of rapamycin (mTOR) pathway has anticancer effect in vivo and in vitro. The aim of this review is to present the anticancer activity of MTF highlighting the importance of the AMPK/mTOR pathway in the cancer process.

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Construction of a ribozyme directed against human interleukin-6 mRNA: evaluation of its catalytic activity in vitro and in vivo

Blood ◽

10.1182/blood.v84.11.3758.bloodjournal84113758 ◽

1994 ◽

Vol 84 (11) ◽

pp. 3758-3765 ◽

Cited By ~ 18

Author(s):

M Mahieu ◽

R Deschuyteneer ◽

D Forget ◽

P Vandenbussche ◽

J Content

Keyword(s):

Interleukin 6 ◽

Ribonuclease Protection Assay ◽

Mammalian Expression ◽

Cell Clones ◽

Ribonuclease Protection ◽

Amniotic Cells ◽

Necrosis Factor

We have designed a ribozyme (Rz) that cleaves human interleukin-6 (IL- 6) mRNA in vivo. This Rz was tested in vitro, and was found to give expected size fragments. It was then incorporated into a mammalian expression vector containing the constitutive cytomegalovirus (CMV) immediate early promoter and transfected into human U amniotic cells (UAC). Cell clones that stably express this catalytic RNA have been obtained. Some of them displayed a marked reduction of tumor necrosis factor (TNF)-induced IL-6 production. Their reduced ability to express IL-6 was related to the amount of Rz they produced and to the extent of IL-6 mRNA cleavage as observed by a ribonuclease protection assay. These data provide a method to study further the role of IL-6 production in various biologic situations, and suggest the feasibility of developing Rzs directed against various cytokines to study their biologic role and mechanism of action.

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