Direct visualization by FRET-FLIM of a putative mechanosome complex involving Src, Pyk2 and MBD2 in living MLO-Y4 cells.
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ABSTRACT:
SUBMITTER: Day RN
PROVIDER: S-EPMC8699642 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mechanical stimulation of the skeleton promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular mechanisms directing anabolic and/or anti-catabolic actions on the skeleton during loading are not fully understood. Identifying molecular mechanisms of mechanotransduction (MTD) signaling cascades could identify new therapeutic targets. Most research into MTD mechanisms is typically focused on understanding the signaling pathways that stimulate new bone formation in response to load. However, we investigated the structural, signaling and transcriptional molecules that suppress the stimulatory effects of loading. The high bone mass phenotype of mice with global deletion of either Pyk2 or Src suggests a role for these tyrosine kinases in repression of bone formation. We used fluid shear stress as a MTD stimulus to identify a novel Pyk2/Src-mediated MTD pathway that represses mechanically-induced bone formation. Our results suggest Pyk2 and Src function as molecular switches that inhibit MTD in our mechanically stimulated osteocyte culture experiments. Once activated by oscillatory fluid shear stress (OFSS), Pyk2 and Src translocate to and accumulate in the nucleus, where they associate with a protein involved in DNA methylation and the interpretation of DNA methylation patterns -methyl-CpG-binding domain protein 2 (MBD2). OFSS-induced Cox-2 and osteopontin expression was enhanced in Pyk2 KO osteoblasts, while inhibition of Src enhanced osteocalcin expression in response to OFSS. We found that Src kinase activity increased in the nucleus of osteocytes in response to OFSS and an interaction activated between Src (Y418) and Pyk2 (Y402) increased in response to OFSS. Thus, as a mechanism to prevent an over-reaction to physical stimulation, mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus that functions to suppress anabolic gene expression.
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Project description:Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.
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Project description:The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLC?-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLC? by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLC? and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLC? on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLC? by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.
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Project description:Membrane compartments function as segregated signaling platforms for different cellular functions. It is not clear how Src is regulated at different membrane compartments. To visualize local Src activity in live cells, a FRET-based Src biosensor was targeted in or outside of lipid rafts at the plasma membrane, via acylation or prenylation modifications on targeting tags either directly fused to the biosensor or coupled to the biosensor through an inducible heterodimerization system. In response to growth factors and pervanadate, the induction of Src activity in rafts was slower and weaker, dependent on actin and possibly its mediated transportation of Src from perinuclear regions to the plasma membrane. In contrast, the induction of Src activity in nonrafts was faster and stronger, dependent on microtubules. Hence, Src activity is differentially regulated via cytoskeleton at different membrane compartments.
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Project description:11?-hydroxysteroid dehydrogenase-2 (11?-HSD2) is one of the key enzymes in glucocorticoid metabolism, which can inactivate local corticosterone and regulate the level of active glucocorticoid in tissues. The expression of 11?-HSD2 and its regulatory pathway serve an important role in the apoptosis of steroid induced osteonecrosis of the femoral head (SANFH). The present study aimed to identify the regulatory effects of cAMP on the expression of Sp1 transcription factor (Sp1) and 11?-HSD2 in osteocytes at the cellular level. Murine long bone osteocyte Y4 (MLO-Y4) clone cells and mouse embryo osteoblast-like (MC3T3-E1) cells were cultured in vitro with adenylate cyclase activator or inhibitor (forskolin and SQ22536, respectively) to investigate the effects of alterations to intracellular cAMP levels. mRNA and protein expression levels of Sp1 and 11?-HSD2 were detected by reverse transcription-quantitative PCR and western blotting, respectively. Compared with the negative control group, the mRNA and protein expression levels of Sp1 were significantly increased in the activation group, whereas Sp1 expression levels were significantly decreased in the inhibition group. Similarly, compared with the negative control group, the mRNA and protein expression levels of 11?-HSD2 were significantly increased in the activator group, but significantly decreased in the inhibitor group. The aforementioned results indicated that intracellular cAMP levels significantly regulated the expression of Sp1 and 11?-HSD2 in mouse osteocytes and osteoblasts. Therefore, the present study suggested a potential therapeutic strategy for the prevention of osteonecrosis of the femoral head.
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Project description:Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain-induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJs) between osteocytes and osteoblasts provides a mechanism whereby flow-induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and prostaglandin E(2) (PGE(2)) increases in response to flow. Unapposed connexons, or hemichannels, are themselves functional and may constitute a novel mechanotransduction mechanism. Using MC3T3-E1 osteoblasts and MLO-Y4 osteocytes, we examined the time course and mechanism of hemichannel activation in response to fluid flow, the composition of the hemichannels, and the role of hemichannels in flow-induced ATP release. We demonstrate that fluid flow activates hemichannels in MLO-Y4, but not MC3T3-E1, through a mechanism involving protein kinase C, which induces ATP and PGE(2) release.
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Project description:Piezo1, a mechanosensitive ion channel, participates in a variety of biological processes in maintaining bone homeostasis. As the most abundant cells in bones of the mammals, osteocytes play an essential role in bone formation, remodeling, and bone mass maintenance. Here, by exposing MLO-Y4 osteocytes to the fluid shear stress (FSS) microenvironment, we explored the effect of Piezo1-mediated FSS on the expression of the molecules critical to the process of bone formation and resorption, Receptor Activator of Nuclear Factor-Kappa-B Ligand (RANKL) and Osteoprotegerin (OPG). It was found that 9 dyne/cm2 loading for 30 minutes showed an upregulation trend on Piezo1 when MLO-Y4 osteocytes were exposed to an FSS microenvironment. FSS promotes the expression of OPG and inhibits the expression of RANKL. The blocker of Piezo1, GsMTx4, downregulates the effect of FSS on the expression of these two molecules. In addition, NOTCH3 was involved in this process. Thus, the results demonstrated that Piezo1-mediated FSS promotes the expression of OPG and inhibits the expression of RANKL via NOTCH3 in MLO-Y4 osteocytes.
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