Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

Bone morphogenetic proteins （Bmp） are well-known to induce bone formation following chondrogenesis, but the direct role of Bmp signaling in the osteoblast lineage is not completely understood. We have recently shown that deletion of the receptor Bmprla in the osteoblast lineage with Dmpl-Cre reduces osteoblast activity in general but stimulates proliferation of preosteoblasts specifically in the cancellous bone region, resulting in diminished periosteal bone growth juxtaposed with excessive cancellous bone formation. Because expression of sclerostin （SOST）, a secreted Wnt antagonist, is notably reduced in the Bmprla- deficient osteocytes, we have genetically tested the hypothesis that increased Wnt signaling might mediate the increase in cancellous bone formation in response to Bmprla deletion. Forced expression of human SOST from a Dmpl promoter fragment partially rescues preosteoblast hyperproliferation and cancellous bone overgrowth in the Bmprla mutant mice, demonstrating functional interaction between Bmp and Wnt signaling in the cancellous bone compat^a-tent. To test whether increased Wnt signaling can compensate for the defect in periosteal growth caused by Bmprla deletion, we have generated compound mutants harboring a hyperactive mutation （A214V） in the Wnt receptor Lrp5. However, the mutant Lrp5 does not restore periosteal bone growth in the Bmprla-deficient mice. Thus, Bmp signaling restricts cancellous bone accrual partly through induction of SOST that limits preosteoblast proliferation, but promotes periosteal bone growth apparently independently of Wnt activation.

Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissuespecific Rbpjk mutant（Prx1Cre;Rbpjkf/f）, Rbpjk mutant/Sox9 haploinsufficient（Prx1Cre;Rbpjkf/f;Sox9f/1）,and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors.

Homeostatic regulation of brain functions by endocannabinoid signaling

Humans have been using Cannabis and its extracts for a few thousand years as a medicinal and recreational drug. How- ever, the chemical component in Cannabis sativa, △9-tet- rahydrocannabinol （△9-THC）, an exogenous cannabinoid, remained unknown until it was isolated and identified as the main psychoactive ingredient （Gaoni and Mechoulam, 1964）.

GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization,mitochondria-targeting and-activating properties of human salivary histatin 1

Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets and activates mitochondria,presenting an important molecular mechanism.However,its regulating signaling pathways remain to be elucidated.We investigated the influence of specific inhibitors of G protein-coupled receptors(GPCR),endocytosis pathways,extracellular signal-regulated kinases1/2(ERK1/2)signaling,p38 signaling,mitochondrial respiration and Na+/K+-ATPase activity on the uptake,mitochondria-targeting and-activating properties of F-Hst1.We performed a si RNA knockdown(KD)to assess the effect of Sigma-2 receptor(S2R)/Transmembrane Protein 97(TMEM97)—a recently identified target protein of Hst1.We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1.Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1.The inhibitors of GPCR,ERK1/2,phagocytosis,and clathrin-mediated endocytosis(CME)as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake,which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity.Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1.We further showed the intracellular trafficking and targeting process of F-Hst1,in which early endosome plays an important role.Overall,phagocytosis,CME,GPCR,ERK signaling,and S2R/TMEM97 are involved in the internalization of Hst1,while only CME and S2R/TMEM97 are critical for its subcellular targeting.The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Promotion of structural plasticity in area V2 of visual cortex prevents against object recognition memory deficits in aging and Alzheimer's disease rodents

Memory deficit,which is often associated with aging and many psychiatric,neurological,and neurodegenerative diseases,has been a challenging issue for treatment.Up till now,all potential drug candidates have failed to produce satisfa ctory effects.Therefore,in the search for a solution,we found that a treatment with the gene corresponding to the RGS14414protein in visual area V2,a brain area connected with brain circuits of the ventral stream and the medial temporal lobe,which is crucial for object recognition memory(ORM),can induce enhancement of ORM.In this study,we demonstrated that the same treatment with RGS14414in visual area V2,which is relatively unaffected in neurodegenerative diseases such as Alzheimer s disease,produced longlasting enhancement of ORM in young animals and prevent ORM deficits in rodent models of aging and Alzheimer’s disease.Furthermore,we found that the prevention of memory deficits was mediated through the upregulation of neuronal arbo rization and spine density,as well as an increase in brain-derived neurotrophic factor(BDNF).A knockdown of BDNF gene in RGS14414-treated aging rats and Alzheimer s disease model mice caused complete loss in the upregulation of neuronal structural plasticity and in the prevention of ORM deficits.These findings suggest that BDNF-mediated neuronal structural plasticity in area V2 is crucial in the prevention of memory deficits in RGS14414-treated rodent models of aging and Alzheimer’s disease.Therefore,our findings of RGS14414gene-mediated activation of neuronal circuits in visual area V2 have therapeutic relevance in the treatment of memory deficits.

RANKL-RANK signaling regulates osteoblast differentiation and bone formation

In the recent two decades, it has been well elucidated that receptor activator of nuclear factor-κB ligand （RANKL; also known as TNFSF11） binding to its receptor RANK （also known as TNFRSF11A） drives osteoclast development as the crucial signaling pathway.;However, accumulating evidence also implies that

YAP promotes osteogenesis and suppresses adipogenic differentiation by regulatingβ-catenin signaling

YAP（yes-associated protein） is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP＇s function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis.YAP is selectively expressed in osteoblast（OB）-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs（bone marrow stromal cells） diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.

Role of chondroitin sulfate proteoglycan signaling in regulating neuroinflammation following spinal cord injury

Spinal cord injury （SCI） elicits a robust inflammatory response that is a hallmark of the secondary injury mechanisms. Neuroinflammation is orchestrated initially by the response of resident astrocytes and microglia to injury, which subsequently facilitates the recruitment of peripheral immune cells into the SCI lesion （Orr and Gensel, 2018）. This inflammatory response contributes to cell death and tissue degeneration through the production of pro-inflammatory cytokines and chemokines, free radicals and proteolytic enzymes. However, neuroinflammatory cells also play beneficial regulatory role in repair mechanisms after SCI by adopting a reparative and wound healing phenotype （Orr and Gensel, 2018; Tran et al., 2018）. Hence, understanding the underlying mechanisms by which immune cells are reg- ulated within the microenvironment of injury would aid in harnessing the reparative potential of inflammation following SCI.

RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation

RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells(BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting β-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs.Interestingly, conditional knockout of rank in BMSCs with Prx1-Cre mice leads to a higher bone mass and increased trabecular bone formation independent of osteoclasts. In addition, rank: Prx1-Cre mice show resistance to ovariectomy-(OVX) induced bone loss. Thus, our results reveal that RANKL signaling regulates both osteoclasts and osteoblasts by inhibition of osteogenic differentiation of BMSCs and promotion of osteoclastogenesis.

Gα_s signaling controls intramembranous ossification during cranial bone development by regulating both Hedgehog and Wnt/β-catenin signaling

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gαs that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia(FD) or progressive osseous heteroplasia(POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gαsregulates Hh signaling in a ligand-independent manner. We further show that Gαscontrols intramembranous ossification by regulating both Hh and Wnt/β-catenin signaling. In addition, Gαsactivation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

Regulated Degradation of HFR1 Desensitizes Light Signaling in Arabidopsis

Arabidopsis seedlings undergo photomorphogenesis in the light and etiolation in the dark. HFR1, a bHLH transcription factor, is required for both phytochrome A (phyA)-mediated far-red and cryptochrome 1 (cry1)-mediated blue light signaling. We report that HFR1 is a short-lived protein in darkness and is degraded through a 26S proteasome-dependent pathway. Light, irrespective of its quality, enhances HFR1 protein accumulation via promoting its stabilization. We demonstrate that HFR1 physically interacts with COP1 and that COP1 exhibits ubiquitin ligase activity toward HFR1 in vitro. In addition, we show that COP1 is required for degradation of HFR1 in vivo. Furthermore, plants overexpressing a C-terminal 161 amino acid fragment of HFR1 (CT161) display enhanced photomorphogenesis, suggesting an autonomous function of CT161 in promoting light signaling. This truncated HFR1 gene product is more stable than the full-length HFR1 protein in darkness, indicating that the COP1-interacting N-terminal portion of HFR1 is essential for COP1-mediated destabilization of HFR1. These results suggest that light enhances HFR1 protein accumulation by abrogating COP1-mediated degradation of HFR1, which is necessary and sufficient for promoting light signaling. Additionally, our results substantiate the E3 ligase activity of COP1 and its critical role in desensitizing light signaling.

Tea domain transcription factor TEAD4 mitigatesTGF-β signaling and hepatocellular carcinomaprogression independently of YAP

Tea domain transcription factor 4 (TEAD4) plays a pivotal role in tissue development and homeostasis by interacting with Yesassociated protein (YAP) in response to Hippo signaling inactivation. TEAD4 and YAP can also cooperate with transforminggrowth factor-β (TGF-β)-activated Smad proteins to regulate gene transcription. Yet, it remains unclear whether TEAD4 playsa YAP-independent role in TGF-β signaling. Here, we unveil a novel tumor suppressive function of TEAD4 in liver cancer viamitigating TGF-β signaling. Ectopic TEAD4 inhibited TGF-β-induced signal transduction, Smad transcriptional activity, and targetgene transcription, consequently suppressing hepatocellular carcinoma cell proliferation and migration in vitro and xenografttumorgrowth in mice. Consistently, depletion of endogenous TEAD4 by siRNAs enhanced TGF-β signaling in cancer cells. Mechanistically,TEAD4 associates with receptor-regulated Smads (Smad2/3) and Smad4 in the nucleus, thereby impairing the binding of Smad2/3to the histone acetyltransferase p300. Intriguingly, these negative effects of TEAD4 on TGF-β/Smad signaling are independent ofYAP, as impairing the TEAD4–YAP interaction through point mutagenesis or depletion of YAP and/or its paralog TAZ has little effect.Together, these results unravel a novel function of TEAD4 in fine tuning TGF-β signaling and liver cancer progression in a YAPindependent manner.

Insights into the regulation of C-repeat binding factors in plant cold signaling

Cold temperatures, a major abiotic stress, threaten the growth and development of plants, worldwide. To cope with this adverse environmental cue, plants from temperate climates have evolved an array of sophisticated mechanisms to acclimate to cold periods, increasing their ability to tolerate freezing stress. Over the last decade, significant progress has been made in determining the molecular mechanisms underpinning cold acclimation, including following the identification of several pivotal components, including candidates for cold sensors, protein kinases, and transcription factors. With these developments, we have a better understanding of the CBF-dependent cold-signaling pathway. In this review, we summarize recent progress made in elucidating the cold-signaling pathways, especially the C-repeat binding factor-dependent pathway, and describe the regulatory function of the crucial components of plant cold signaling. We also discuss the unsolved questions that should be the focus of future work.

Expression of Extracellular Signal-regulated Kinase and Angiotensin-converting Enzyme in Human Atria during Atrial Fibrillation

In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2 mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P <0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.

Magnaporthe Rab5 homologs show distinct functions in nerve growth factor(NGF)-mediated neurite outgrowth and cell differentiation

Nerve growth factor（NGF） binds to TrkA and forms a NGF/TrkA complex at the cell surface,which is then internalized into signaling endosomes and promotes neuronal survival and neurite outgrowth.The small GTPase Rab5 is reported to localize on the plasma membrane and early endosomes,regulating endosome fusion.It was reported that endogenous Rab5 function may need to be suppressed during NGF-induced neurite outgrowth and cell differentiation.Two Rab5 homologs（MoRab5A：MGG_06241 and MoRab5B：MGG_01185） were characterized from the rice blast fungus Magnaporthe oryzae,and MoRab5 B was identified as the Rab5 ortholog promoting early endosomal fusion,while MoRab5 A specialized to perform a non-redundant function in endosomal sorting.In this study,we examined whether MoRab5 A and MoRab5 B play different roles in NGF-induced neurite outgrowth and cell differentiation in PC12 cells（a rat pheochromocytoma cell line）.Our data showed that MoRab5 B is a negative regulator of NGF signaling and neurite outgrowth in PC12 cells,similar to human Rab5（hRab5）.MoRab5B：WT inhibits NGF signaling-dependent neurite outgrowth while the dominant-negative MoRab5 B mutant（MoRab5B：DN） enhances NGF signaling and neurite outgrowth.In contrast,MoRab5A：WT and MoRab5A：DN both significantly promote NGF-induced neurite outgrowth,indicating that MoRab5 B is more similar to hRab5 than MoRab5 A in the regulation of NGF signal transduction.

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embryonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140（RIP140） promotes neuronal differentiation of N2 a cells via extracellular regulated kinase 1/2（ERK1/2） signaling.However,involvement of RIP140 in human neural differentiation remains unclear.We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells.Moreover,RIP140 negatively correlated with stem cell markers Oct4 and Sox2 during early stages of neural differentiation,and positively correlated with the neural stem cell marker Nestin during later stages.Thus,ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.

Signaling control of the constitutive androstane receptor （CAR）

The constitutive androstane receptor （CAR, NRll3） plays a crucial role in the regulation of drug metabolism, energy homeostasis, and cancer development through modulating the transcription of its numerous target genes. Different from prototypical nuclear receptors, CAR can be activated by either direct ligand binding or ligand-independent （indirect） mechanisms both initiated with nuclear translocation of CAR from the cytoplasm. In comparison to the well-defined ligand-based activation, indirect activation of CAR appears to be exclusively involved in the nuclear translocation through mecha- nisms yet to be fully understood. Accumulating evi- dence reveals that without activation, CAR forms a protein complex in the cytoplasm where it can be func- tionally affected by multiple signaling pathways. In this review, we discuss recent progresses in our under- standing of the signaling regulation of CAR nuclear accumulation and activation. We expect that this review will also provide greater insight into the similarity and difference between the mechanisms of direct vs. indirect human CAR activation.

Molecular Mechanisms Regulating Rapid Stress Signaling Networks in Arabidopsis

As sessile organisms plants must ronmental conditions. To survive cope with ever changing enviplants have evolved elaborate mechanisms to perceive and rapidly respond to a diverse range of abiotic and biotic stresses. Central to this response is the ability to modulate gene expression at both the transcriptional and posttranscriptional levels. This review will focus on recent progress that has been made towards understanding the rapid reprogramming of the transcriptome that occurs in response to stress as well as emerging mechanisms underpinning the reprogramming of gene expression in response to stress,

Arabidopsis small ubiquitin-related modifier protease ASP1 positively regulates abscisic acid signaling during early seedling development~~

The small ubiquitin-related modifier （SUMO） modification plays an important role in the regulation of abscisic acid （ABA） signaling, but the function of the SUMO protease, in ABA signaling, remains largely unknown. Here, we show that the SUMO protease, ASPI positively regulates ABA signaling. Mutations in ASPI resulted in an ABA-insensitive phenotype, during early seedling develop- ment. Wild-type ASP1 successfully rescued, whereas an ASPI mutant （C577S）, defective in SUMO protease activity, failed to rescue, the ABA-insensitive phenotype of asp1-1. Expression of ABI5 and MYB3o target genes was attenuated in asp^-I and our genetic analyses revealed that ASP1 may function upstream of ABI5 and MYB3o.

Activation of glycine site and GluN2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats:Implications for affective pain

Objective The rostral anterior cingulate cortex （rACC） is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors （NMDARs） are highly expressed in the rACC and mediate painrelated affect by activating a signaling pathway that involves cyclic adenosine monophosphate （cAMP）/protein ki- nase A （PKA） and/or extracellular regulated kinase （ERK）/cAMP-response element-binding protein （CREB）. The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC. Methods Immunohistochemistry and Western blot analy- sis were used to separately assess the expression of phospho-ERK （pERK） and phospho-CREB （pCREB） in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB. Results Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antago- nist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine- binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up- regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB. Conclusion Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.