Fish bone peptide promotes osteogenic differentiation of MC3T3‐E1 pre‐osteoblasts through upregulation of MAPKs and Smad pathways activated BMP‐2 receptor

Fish bone, a by‐product of fishery processing, is composed of protein, calcium, and other minerals. The objective of this study was to investigate the effects of a bioactive peptide isolated from the bone of the marine fish, Johnius belengerii, on the osteoblastic differentiation of MC3T3‐E1 pre‐osteoblasts. Post consecutive purification by liquid chromatography, a potent osteogenic peptide, composed of 3 amino acids, Lys‐Ser‐Ala (KSA, MW: 304.17 Da), was identified. The purified peptide promoted cell proliferation, alkaline phosphatase activity, mineral deposition, and expression levels of phenotypic markers of osteoblastic differentiation in MC3T3‐E1 pre‐osteoblast. The purified peptide induced phosphorylation of mitogen‐activated protein kinases, including p38 mitogen‐activated protein kinase, extracellular regulated kinase, and c‐Jun N‐terminal kinase as well as Smads. As attested by molecular modelling study, the purified peptide interacted with the core interface residues in bone morphogenetic protein receptors with high affinity. Thus, the purified peptide could serve as a potential pharmacological substance for controlling bone metabolism.     

Gypenoside inhibits interleukin‐1β‐induced inflammatory response in human osteoarthritis chondrocytes

Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti‐inflammation, anti‐oxidation, and anti‐tumor. However, the effects of GP on IL‐1β‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti‐inflammatory effects of GP on IL‐1β‐stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose‐dependently inhibited IL‐1β‐induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL‐1β. Finally, we found that pretreatment of GP obviously suppressed NF‐κB activation in IL‐1β‐stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro‐protective effects, at least in part, through inhibiting the activation of NF‐κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients.     

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor‐like kinases. The symbiotic receptor‐like kinases nodulation receptor‐like kinase (NORK) and lysin motif domain‐containing receptor‐like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor‐like kinases, very little is known about their phosphorylation substrates. Using this high‐throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor‐like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high‐throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.     

Tau‐induced upregulation of C/EBPβ‐TRPC1‐SOCE signaling aggravates tauopathies: A vicious cycle in Alzheimer neurodegeneration

Intracellular accumulating of the hyperphosphorylated tau plays a pivotal role in neurodegeneration of Alzheimer disease (AD), but the mechanisms underlying the gradually aggravated tau hyperphosphorylation remain elusive. Here, we show that increasing intracellular tau could upregulate mRNA and protein levels of TRPC1 (transient receptor potential channel 1) with an activated store‐operated calcium entry (SOCE), an increased intraneuronal steady‐state [Ca²⁺]_i, an enhanced endoplasmic reticulum (ER) stress, an imbalanced protein kinases and phosphatase, and an aggravated tauopathy. Furthermore, overexpressing TRPC1 induced ER stress, kinases‐phosphatase imbalance, tau hyperphosphorylation and cognitive deficits in cultured neurons and mice, while pharmacological inhibiting or knockout TRPC1 attenuated the hTau‐induced deregulations in SOCE, ER homeostasis, kinases‐phosphatase balance, and tau phosphorylation level with improved synaptic and cognitive functions. Finally, an increased CCAAT‐enhancer‐binding protein (C/EBPβ) activity was observed in hTau‐overexpressing cells and the hippocampus of the AD patients, while downregulating C/EBPβ by siRNA abolished the hTau‐induced TRPC1 upregulation. These data reveal that increasing intracellular tau can upregulate C/EBPβ‐TRPC1‐SOCE signaling and thus disrupt phosphorylating system, which together aggravates tau pathologies leading to a chronic neurodegeneration.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

Both W9 and OP3‐4 were known to bind the receptor activator of NF‐κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide‐induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3‐4 accelerated BMP‐2‐induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL‐binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP‐2‐induced bone regeneration by the RANKL‐binding peptides.     

Kinase-interacting substrate screening is a novel method to identify kinase substrates

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.     

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 β/β‐catenin pathways

Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre‐existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow‐derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3β and entry of β‐catenin into the nucleus. Imperatorin also enhanced the production of phospho‐AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3β. We used ipatasertib, a pan‐AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3β/β‐catenin pathway. Next, we used rat bone marrow‐derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3β/β‐catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.     

Trolox‐induced cardiac differentiation is mediated by the inhibition of Wnt/β‐catenin signaling in human embryonic stem cells

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/β‐catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6‐Hydroxy‐2,5,7,8‐tetramethylchromane‐2‐carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time‐ and dose‐dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/β‐catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/β‐catenin regulator.     

Lanthanum suppresses osteoblastic differentiation via pertussis toxin‐sensitive G protein signaling in rat vascular smooth muscle cells

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast‐like cells. After demonstrating that lanthanum chloride (LaCl₃) suppresses hydrogen peroxide‐enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccol's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM β‐glycerophosphate (β‐GP) in the presence or absence of LaCl₃. Exposure of cells to LaCl₃ suppressed the β‐GP‐induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM α‐actin. Furthermore, LaCl₃ activated the phosphorylation of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl₃. In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein‐mediated signaling pathway, repealed all the changes induced by LaCl₃. These findings demonstrate that LaCl₃ suppresses the β‐GP‐induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx‐sensitive G proteins. J. Cell. Biochem. 108: 1184–1191, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

Construction of human activity‐based phosphorylation networks

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.     

A chimeric mechanism for polyvalent trans‐phosphorylation of PKA by PDK1

Phosphorylation on the activation loop of AGC kinases is typically mediated by PDK1. The precise mechanism for this in‐trans phosphorylation is unknown; however, docking of a hydrophobic (HF) motif in the C‐tail of the substrate kinase onto the N‐lobe of PDK1 is likely an essential step. Using a peptide array of PKA to identify other PDK1‐interacting sites, we discovered a second AGC‐conserved motif in the C‐tail that interacts with PDK1. Since this motif [FD(X)_1‐2Y/F] lies in the active site tether region and in PKA contributes to ATP binding, we call it the Adenosine binding (Ade) motif. The Ade motif is conserved as a PDK1‐interacting site in Akt and PRK2, and we predict it will be a PDK1‐interacting site for most AGC kinases. In PKA, the HF motif is only recognized when the turn motif Ser338 is phosphorylated, possibly serving as a phosphorylation “switch” that regulates how the Ade and HF motifs interact with PDK1. These results demonstrate that the extended AGC C‐tail serves as a polyvalent element that trans‐regulates PDK1 for catalysis. Modeling of the PKA C‐tail onto PDK1 structure creates two chimeric sites; the ATP binding pocket, which is completed by the Ade motif, and the C‐helix, which is positioned by the HF motif. Together, they demonstrate substrate‐assisted catalysis involving two kinases that have co‐evolved as symbiotic partners. The highly regulated turn motifs are the most variable part of the AGC C‐tail. Elucidating the highly regulated cis and trans functions of the AGC tail is a significant future challenge.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

p21‐activated kinase 7 is an oncogene in human osteosarcoma

p21‐activated kinase 7 (PAK7), also named as PAK5, is a member of Rac/Cdc42‐associated Ser/Thr protein kinases. It is overexpressed in some types of cancer such as colorectal and pancreatic cancers. However, the expression status and biological function of PAK7 in osteosarcoma are still ambiguous. To evaluate the expression levels of PAK7 in osteosarcoma tissues and cell lines, immunohistochemistry was used. To investigate the role of PAK7 in cell proliferation, apoptosis and tumorigenicity in vitro and vivo, a recombinant lentivirus expressing PAK7 short hairpin RNA (Lv‐shPAK7) was developed and transfected into Saos‐2 cells. The silencing effect of PAK7 was confirmed by quantitative real‐time PCR (qRT‐PCR) and Western blot technique. PAK7 was overexpressed in osteosarcoma tissue and cell line. By knocking‐down of PAK7, the proliferation and colony formation of Saos‐2 cells were inhibited and apoptosis enhanced significantly. The in vivo tumorigenic ability in xenograft model of Saos‐2 cells was also notably inhibited when PAK7 was knocked down. Our results imply that PAK7 promotes cell proliferation and tumorigenesis and may be an attractive candidate for the therapeutic target of osteosarcoma.