MEK5-ERK5 Axis Promotes Self-renewal and Tumorigenicity of Glioma Stem Cells.

Glioma stem cells (GSC) promote the malignancy of glioblastoma (GBM), the most lethal brain tumor. ERK5 belongs to the MAPK family. Here, we demonstrated that MAPK kinase 5 (MEK5)-ERK5-STAT3 pathway plays an essential role in maintaining GSC stemness and tumorigenicity by integrating genetic and pharmacologic manipulation and RNA sequencing analysis of clinical specimens. ERK5 was highly expressed and activated in GSCs. ERK5 silencing by short hairpin RNA in GSCs suppressed the self-renewal potential and GBM malignant growth concomitant with downregulation of STAT3 phosphorylation. Conversely, the activation of the MEK5-ERK5 pathway by introducing ERK5 or MEK5 resulted in increased GSC stemness. The introduction of STAT3 counteracted the GSC phenotypes by ERK5 silencing. Moreover, ERK5 expression and signaling are associated with poor prognosis in patients with GBM with high stem cell properties. Finally, pharmacologic inhibition of ERK5 significantly inhibited GSC self-renewal and GBM growth. Collectively, these findings uncover a crucial role of the MEK5-ERK5-STAT3 pathway in maintaining GSC phenotypes and GBM malignant growth, thereby providing a potential target for GSC-directed therapy. Significance: In this study, we demonstrated that MEK5-ERK5-STAT3 axis plays a critical role in maintaining stemness and tumorigenicity in GSCs by using genetic, pharmacologic, and bioinformatics tools, identifying the MEK5-ERK5-STAT3 axis as a potential target for GSC-directed therapy.

l-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis.

Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

Amelioration of Osteoarthritis Development by Daily Oral Supplementation of Royal Jelly.

Royal jelly (RJ), an essential food for the queen honeybee, has a variety of biological activities. Although RJ exerts preventive effects on various lifestyle-related diseases, such as osteoporosis and obesity, no study evaluated the effect of RJ on the development of osteoarthritis (OA), the most common degenerative joint disease. Here, we showed that daily oral administration of raw RJ significantly prevented OA development in vivo following surgically-induced knee joint instability in mice. Furthermore, in vitro experiments using chondrocytes, revealed that raw RJ significantly reduced the expression of inflammatory cytokines and enzymes critical for the degradation of the extracellular matrix (ECM). Similar results were observed after treatment with 10-hydroxy-2-decenoic acid, the most abundant and unique fatty acid in raw RJ. Our results suggest that oral supplementation with RJ would benefit the maintenance of joint health and prophylaxis against OA, possibly by suppressing the activity of inflammatory cytokines and ECM-degrading enzymes.

Conditional inactivation of the L-type amino acid transporter LAT1 in chondrocytes models idiopathic scoliosis in mice.

Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathway but inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.

The role of CDK8 in mesenchymal stem cells in controlling osteoclastogenesis and bone homeostasis.

Bone marrow mesenchymal stem cells (MSCs) are critical regulators of postnatal bone homeostasis. Osteoporosis is characterized by bone volume and strength deterioration, partly due to MSC dysfunction. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Here, CDK8 in MSCs was identified as important for bone homeostasis. CDK8 level was increased in aged MSCs along with the association with aging-related signals. Mouse genetic studies revealed that CDK8 in MSCs plays a crucial role in bone resorption and homeostasis. Mechanistically, CDK8 in MSCs extrinsically controls osteoclastogenesis through the signal transducer and transcription 1 (STAT1)-receptor activator of the nuclear factor κ Β ligand (RANKL) axis. Moreover, aged MSCs have high osteoclastogenesis-supporting activity, partly through a CDK8-dependent manner. Finally, pharmacological inhibition of CDK8 effectively repressed MSC-dependent osteoclastogenesis and prevented ovariectomy-induced osteoclastic activation and bone loss. These findings highlight that the CDK8-STAT1-RANKL axis in MSCs could play a crucial role in bone resorption and homeostasis.

Erk5 in Bone Marrow Mesenchymal Stem Cells Regulates Bone Homeostasis by Preventing Osteogenesis in Adulthood.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

SMURF2 phosphorylation at Thr249 modifies glioma stemness and tumorigenicity by regulating TGF-ß receptor stability.

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorigenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorigenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-ß receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

CDK8 maintains stemness and tumorigenicity of glioma stem cells by regulating the c-MYC pathway.

Glioblastoma (GBM) is the most malignant form of glioma. Glioma stem cells (GSCs) contribute to the initiation, progression, and recurrence of GBM as a result of their self-renewal potential and tumorigenicity. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in gliomagenesis remain largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in GSCs. The genetic inhibition of CDK8 by shRNA or CRISPR interference resulted in an abrogation of the self-renewal potential and tumorigenicity of patient-derived GSCs, which could be significantly rescued by the ectopic expression of c-MYC, a stem cell transcription factor. Moreover, we demonstrated that the pharmacological inhibition of CDK8 significantly attenuated the self-renewal potential and tumorigenicity of GSCs. CDK8 expression was significantly higher in human GBM tissues than in normal brain tissues, and its expression was positively correlated with stem cell markers including c-MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. Collectively, these findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs; these findings also identify the CDK8-c-MYC axis as a potential target for GSC-directed therapy.

Daily oral supplementation of Hochu-Ekki-To prevents osteoclastic activation and bone loss in ovariectomized mice.

Bone remodeling is sophisticatedly regulated by two different cell types: bone-resorbing osteoclasts and bone-forming osteoblasts. Hochu-Ekki-To, a Japanese traditional herbal medicine, is commonly used for the treatment of chronic diseases or frailty after an illness; however, its effects on metabolic bone diseases such as osteoporosis are not well known. We herein report that daily oral Hochu-Ekki-To administration significantly inhibits osteoclast activation as well as the reduction in bone volume in ovariectomized mice. Our results suggest that supplementation with Hochu-Ekki-To might be beneficial for the prophylaxis and treatment of metabolic bone diseases associated with abnormal osteoclast activation.

mTORC1 Overactivation Leads to Abnormalities in Skeletal Development.

The mechanistic/mammalian target of rapamycin complex-1 (mTORC1) integrates multiple signaling pathways and regulates various cellular processes. Tuberous sclerosis complex 1 (Tsc1) and complex 2 (Tsc2) are critical negative regulators of mTORC1. Mouse genetic studies, including ours, have revealed that inactivation of mTORC1 in undifferentiated mesenchymal cells and chondrocytes leads to severe skeletal abnormalities, indicating a pivotal role for mTORC1 in skeletogenesis. Here, we show that hyperactivation of mTORC1 influences skeletal development through its expression in undifferentiated mesenchymal cells at the embryonic stage. Inactivation of Tsc1 in undifferentiated mesenchymal cells by paired-related homeobox 1 (Prx1)-Cre-mediated recombination led to skeletal abnormalities in appendicular skeletons. In contrast, Tsc1 deletion in chondrocytes using collagen type II α1 (Col2a1)-Cre or in osteoprogenitors using Osterix (Osx)-Cre did not result in skeletal defects in either appendicular or axial skeletons. These findings indicate that Tsc complex-mediated chronic overactivation of mTORC1 influences skeletal development at the embryonic stage through its expression in undifferentiated mesenchymal cells but not in chondrocytes or osteoprogenitors.

mTOR regulates skeletogenesis through canonical and noncanonical pathways.

The mechanistic/mammalian target of rapamycin (mTOR) regulates various cellular processes, in part through incorporation into distinct protein complexes. The mTOR complex 1 (mTORC1) contains the Raptor subunit, while mTORC2 specifically contains the Rictor subunit. Mouse genetic studies, including ours, have revealed a critical role for mTOR in skeletogenesis through its expression in undifferentiated mesenchymal cells. In addition, we have recently revealed that mTORC1 expression in chondrocytes is crucial for skeletogenesis. Recent work indicates that mTOR regulates cellular functions, depending on the context, through both complex-dependent (canonical pathway) and complex-independent roles (noncanonical pathway). Here, we determined that mTOR regulates skeletal development through the noncanonical pathway, as well as the canonical pathway, in a cell-type and context-specific manner. Inactivation of Mtor in undifferentiated mesenchymal cells or chondrocytes led to either severe hypoplasia in appendicular skeletons or a severe and generalized chondrodysplasia, respectively. Moreover, Rictor deletion in undifferentiated mesenchymal cells or chondrocytes led to mineralization defects in some skeletal components. Finally, we revealed that simultaneous deletion of Raptor and Rictor in undifferentiated mesenchymal cells recapitulated the appendicular skeletal phenotypes of Mtor deficiency, whereas chondrocyte-specific Raptor and Rictor double-mutants exhibited milder hypoplasia of appendicular and axial skeletons than those seen upon Mtor deletion. These findings indicate that mTOR regulates skeletal development mainly through the canonical pathway in undifferentiated mesenchymal cells, but at least in part through the noncanonical pathway in chondrocytes.