Zhipeng Zou

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-κB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Elevation of n-3/n-6 PUFAs ratio suppresses mTORC1 and prevents colorectal carcinogenesis associated with APC mutation.

Although epidemiological and preclinical studies have shown the preventative effect of n-3 polyunsaturated fatty acids (PUFAs) on colorectal cancer (CRC), the underlying molecular mechanisms are not clear. In this study, we revealed that elevation of n−3/n-6 PUFAs ratio suppress the mechanistic target of rapamycin complex 1 (mTORC1) and prevent colorectal tumorigenesis. The transgenic expression of fat-1, a desaturase that catalyzes the conversion of n-6 to n-3 PUFAs and produces n-3 PUFAs endogenously, repressed colorectal tumor cell growth and remarkably reduced tumor burden, and alleviated anemia as well as hyperlipidemia in APCMin/+ (adenomatous polyposis coli) mice, a classic CRC model that best simulates most clinical cases. In contrast to arachidonic acid (AA, C20:4 n−6), either Docosahexaenoic acid (DHA, C22:6 n−3), eicosapentaenoic acid (EPA, C20:5 n−3), or a combination of DHA and AA, efficiently inhibited the proliferation of CRC cell lines and promoted apoptosis in these cells. The ectopic expression of fat-1 had similar effects in colon epithelial cells with APC depletion. Mechanistically, elevation of n−3/n−6 ratio suppressed mTORC1 activity in tumors of APCMin/+ mice, CRC cell lines with APC mutation, and in normal colon epithelial cells with APC depletion. In addition, elevation of n−3/n−6 ratio repressed mTORC1 activity and inhibited adipogenic differentiation in preadipocytes with APC knockdown, as well as alleviated hyperlipidemia in APCMin/+ mice. Taken together, our findings have provided novel insights into the potential mechanism by which increase in n−3/n−6 PUFAs ratio represses CRC development, and also a new rationale for utilizing n-3 PUFAs in CRC prevention and treatment.

Targeted Inhibition of Rictor/mTORC2 in Cancer Treatment: A New Era after Rapamycin.

The evolutionarily conserved mechanistic target of rapamycin (mTOR) forms two functionally distinct complexes, mTORC1 and mTORC2. mTORC1, consisting of mTOR, raptor, and mLST8 (GβL), is sensitive to rapamycin and thought to control autonomous cell growth in response to nutrient availability and growth factors. mTORC2, containing the core components mTOR, mLST8, Rictor, mSIN1, and Protor1/2 is largely insensitive to rapamycin. mTORC2 specifically senses growth factors and regulates cell proliferation, metabolism, actin rearrangement, and survival. Dysregulation of mTOR signaling often occurs in a variety of human malignant diseases, rendering it a crucial and validated target in cancer treatment. However, the effectiveness of rapamycin as single-agent therapy is suppressed, in part, by the numerous strong mTORC1-dependent negative feedback loops. Although preclinical and clinical studies of ATP-competitive mTOR inhibitors that target both mTORC1 and mTORC2 have shown greater effectiveness than rapalogs for cancer treatment, the mTORC1 inhibition-induced negative feedback activation of PI3- K/PDK1 and Akt (Thr308) may be sufficient to promote cell survival. Recent cancer biology studies indicated that mTORC2 is a promising target, since its activity is essential for the development of a number of cancers. These studies provide a rationale for developing inhibitors specifically targeting mTORC2, which do not perturb the mTORC1- dependent negative feedback loops and have a more acceptable therapeutic window. This review summarizes the present understanding of mTORC2 signaling and functions, especially tumorigenic functions, highlighting the current status and future perspectives for targeting mTORC2 in cancer treatment.

mTORC1 Activation Promotes Spermatogonial Differentiation and Causes Subfertility in Mice

ABSTRACT Spermatogenesis is a continuous process, relying on the proliferation and differentiation of spermatogonia. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, proliferation, and differentiation, yet its roles in the regulation of spermatogonial development and differentiation remain unclear. Here, we found that spermatogonia display stage-dependent mTORC1 activity during their postnatal development, with extremely low activity in undifferentiated spermatogonia and high activity in differentiated spermatogonia. To examine this difference, we generated mutant mice with activated mTORC1 in a subset of undifferentiated spermatogonia by conditionally deleting the mTORC1 inhibitor TSC1. The knockout mice demonstrated testicular developmental defects, partial spermatogenic arrest, excessive germ cell loss, sperm count reduction, and subfertility. Importantly, mTORC1 activation promoted spermatogonial differentiation at the expense of germline maintenance, inducing the early depletion of germ cells, and thus impairing spermatogenesis. In summary, our study defines the critical roles of mTORC1 in the maintenance of the spermatogonial population and functions.

Osteoblasts support megakaryopoiesis through production of interleukin 9

Severe thrombocytopenia is a significant challenge in patients undergoing myelosuppressive chemotherapy for malignancies. Understanding the biology of platelet-producing megakaryocytes development in the bone marrow microenvironment may facilitate the development of novel therapies to stimulate platelet production and prevent thrombocytopenia. We report here that osteoblasts supported megakaryopoiesis by secreting interleukin-9 (IL-9), which stimulated IL-9 receptor (IL-9R)/Stat3 signaling in promoting megakaryopoiesis. IL-9 production in osteoblasts was negatively regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signaling in a NF-κB-dependent manner. Constitutive activation of mTORC1 inhibited IL-9 production in osteoblasts and suppressed megakaryocytic cells expansion, whereas mTORC1 inactivation increased IL-9 production and enhanced megakaryocyte and platelet numbers in mice. In mouse models, we showed that IL-9 administration stimulated megakaryopoiesis, whereas neutralizing endogenous IL-9 or IL-9R depletion inhibited the process. Importantly, we found that low doses of IL-9 efficiently prevented chemotherapy-induced thrombocytopenia (CIT) and accelerated platelet recovery after CIT. These data indicate that IL-9 is an essential regulator of megakaryopoiesis and a promising therapeutic agent for treatment of thrombocytopenia such as CIT.

Neuronal mTORC1 is required for maintaining the nonreactive state of astrocytes

Astrocytes respond to CNS insults through reactive astrogliosis, but the underlying mechanisms are unclear. In this study, we show that inactivation of mechanistic target of rapamycin complex (mTORC1) signaling in postnatal neurons induces reactive astrogliosis in mice. Ablation of Raptor (an mTORC1-specific component) in postmitotic neurons abolished mTORC1 activity and produced neurons with smaller soma and fewer dendrites, resulting in microcephaly and aberrant behavior in adult mice. Interestingly, extensive astrogliosis without significant astrocyte proliferation and glial scar formation was observed in these mice. The inhibition of neuronal mTORC1 may activate astrogliosis by reducing neuron-derived fibroblast growth factor 2 (FGF-2), which might trigger FGF receptor signaling in astrocytes to maintain their nonreactive state, and FGF-2 injection successfully prevented astrogliosis in Raptor knock-out mice. This study demonstrates that neuronal mTORC1 inhibits reactive astrogliosis and plays an important role in CNS pathologies.

Inactivation of mTORC1 Signaling in Osterix-Expressing Cells Impairs B-cell Differentiation: mTORC1 IN OSX-EXPRESSING CELLS REGULATES B-CELL DEVELOPMENT

Osteoblasts provide a microenvironmental niche for B‐cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B‐cell development. Inactivation of mTORC1 in osterix‐expressing cells (mainly osteoblast lineage) through Osx‐Cre‐directed deletion of Raptor (an mTORC1‐specific component) resulted in a reduction in the total B‐cell population in the BM, which was due to a block in early B‐cell development from the pro‐B to pre‐B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin‐7 (IL‐7) expression in osterix‐expressing immature osteoblasts, which caused the abnormality of IL‐7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre‐B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL‐7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti‐IL‐7 neutralizing antibody failed to differentiate into pre‐B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B‐cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B‐cell differentiation via regulating IL‐7 expression.

Colonic epithelial mTORC1 promotes ulcerative colitis through COX-2-mediated Th17 responses

The functional role of colonic epithelium in the pathogenesis of ulcerative colitis (UC) remains unclear. Here, we reveal a novel mechanism by which colonic epithelia recruit T helper-17 (Th17) cells during the onset of UC. mTOR complex 1 (mTORC1) was hyper-activated in colonic epithelia of UC mice. While colonic epithelial TSC1 (mTORC1 negative regulator) disruption induced constitutive mTORC1 activation in the colon epithelia and aggravated UC, RPTOR (essential mTORC1 component) depletion inactivated mTORC1 and ameliorated UC. TSC1 deficiency enhanced, whereas RPTOR ablation reduced the expression of cyclooxygenase 2 (COX-2), interleukin-1 (IL-1), IL-6, and IL-23, as well as Th17 infiltration in the colon. Importantly, inhibition of COX-2 reversed the elevation in the expression of these proinflammatory mediators induced by TSC1 deficiency, and subsequently reduced the symptoms and pathological characteristics of UC in mouse models. Mechanistically, mTORC1 activates COX-2 transcription via phosphorylating STAT3 and enhancing it’s binding to the COX-2 promoter. Consistently, enhanced mTORC1 activity and COX2 expression, as well as strong positive correlation between each other, were observed in colonic epithelial tissues of UC patients. Collectively, our study demonstrates an essential role of epithelial mTORC1 in UC pathogenesis and establishes a novel link between colonic epithelium, Th17 responses, and UC development.

Loss of DEPTOR in renal tubules protects against cisplatin-induced acute kidney injury

DEP domain containing mTOR-interacting protein (DEPTOR) was originally identified as an in vivo dual inhibitor of mechanistic target of rapamycin (mTOR). It was recently reported to be involved in renal physiology and pathology in vitro; however, its detailed roles and mechanisms in vivo are completely unknown. We observed that DEPTOR expression in the kidney was markedly increased on day 3 after cisplatin treatment, at which time cell apoptosis peaked, implicating DEPTOR in cisplatin-induced acute kidney injury (AKI). We then used the Cre–LoxP system to generate mutant mice in which the DEPTOR gene was specifically deleted in the proximal tubule cells. DEPTOR deficiency did not alter the renal histology or functions in the saline-treated group, indicating that DEPTOR is not essential for kidney function under physiological conditions. Interestingly, DEPTOR deletion extensively preserved the renal histology and maintained the kidney functions after cisplatin treatment, suggesting that the absence of DEPTOR ameliorates cisplatin-induced AKI. Mechanistically, DEPTOR modulated p38 MAPK signaling and TNFα production in vivo and in vitro, rather than mTOR signaling, thus moderating the inflammatory response and cell apoptosis induced by cisplatin. Collectively, our findings demonstrate the roles and mechanisms of DEPTOR in the regulation of the renal physiology and pathology, and demonstrate that the loss of DEPTOR in the proximal tubules protects against cisplatin-induced AKI.

Raptor directs Sertoli cell cytoskeletal organization and polarity in the mouse testis

Abstract Sertoli cells (SCs) play a central role in testis development, and their normal number and functions are required for spermatogenesis. Although the canonical tuberous sclerosis complex–mammalian target of rapamycin complex 1(TSC-mTORC1) pathway is critical for testis development and spermatogenesis, the signaling mechanisms governing SC functions remain unclear. In this study,we generated two SC-specific mouse mutants using the Cre–LoxP system. Loss of Raptor (a key component of mTORC1) caused severe tubular degeneration in the neonatal testis and adult mice displayed azoospermia, while adult Rheb (an upstream activator for mTORC1) mutant mice had intact tubules and many sperm in their epididymides. Disruption of cytoskeletal organization, including actin, microtubules, and SC-intrinsic vimentin, was observed in Raptor but not Rheb mutant mice. We investigated the reasons for these different effects by whole-transcriptome sequencing, and found that expression of the tight junction adaptor protein cingulin was significantly reduced in Raptor mutant mice. The expression profile of cingulin was synchronous with the differentiation and cytoskeletal dynamics of SCs in control mice, but was disordered in Raptor mutant mice. Furthermore, activity of the small GTPase Rac1 was reduced and expression of the guanine exchange factor for Rac1, Asef, was decreased in Raptor but not Rheb mutant mice. Collectively, these findings establish novel functions of Raptor, independent of the canonical Rheb/mTORC1 pathway, in controlling cytoskeletal homeostasis and cell polarity in SCs, by affecting cingulin expression and Rac1 activity. Summary Sentence Raptor has individual functions independence of canonical Rheb/mTOR signaling to regulate cytoskeletal dynamics and cell polarity in Sertoli cells by affecting Cingulin expression and Rac1 activity.