Estrogen Differentially Regulates Protein Abundance in Exosomes Released From Immortalized Kisspeptin Neurons in Vitro

Abstract Estrogen (E2) is essential for multiple physiological effects in females, ensuring maximum reproductive fitness and maintaining skeletal homeostasis. E2 has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), an effect widely accepted to be mediated directly at bone. A recent landmark study (Herber et al., Nat Commun 2019) demonstrated bone density increases in female mice harboring ERα-deletions specifically in arcuate Kiss-1 neurons. In this study, bone from transgenic females showed higher osteoblast functioning and increases in the expression of sp7 and runx2, positing a direct neural-bone regulatory axis altered by circulating E2 acting in brain. Our laboratory has used two immortalized Kisspeptin (Kiss1)-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) as models to explore the role of Kiss-1 in multiple physiological regulatory contexts. We recently determined that factors in the media of female ARC-derived KTaR-1 cells can affect parameters of osteoblast function in vitro, including increases in sp7 and runx2 expression, and formation of bone matrix (evaluated by Alizarin Red assay). Exposure of canine osteosarcoma cells to conditioned media from KTaR-1 cells led to increases in sp7 expression in an E2-dependent manner, and 24h E2-deprivation of these neurons stimulated secretion of osteogenic factors. In this current study, we have used LCMS-MS proteomic analysis to determine the contents of exosomes isolated from Kisspeptin neurons under varying E2 exposure conditions in vitro. Preliminary results reveal ~150-170 proteins up-regulated by E2 exposure and ~200-220 proteins downregulated by E2 exposure in exosomes of both KTaR-1 and KTaV-3 Kisspeptin neurons. Estrogen-regulated Kiss-1 exosomal proteins include several candidates involved in bone remodeling (pentraxin, osteonectin, osteoclast-stimulating factor-1) and neuronal synaptic plasticity and signaling (annexins, semaphorins, connexins). Current work is exploring the effects of exposure of purified exosomes on morphology and gene expression in immortalized GnRH neurons and osteoblasts. While further study is required, initial results suggest that exosomes may represent additional cellular communication pathways utilized by Kisspeptin neurons to elicit changes in brain and bone.

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SUN-263 In Vitro Modeling of Brain-Bone Communication: Factors Secreted from Immortalized Female Kisspeptin Neurons Modulate Gene Expression in Osteoblasts

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1925 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Teagan James ◽

Marcus Weinman ◽

Patrick Everett Chappell

Keyword(s):

Estrogen Receptor Alpha ◽

Bone Matrix ◽

In Vitro Models ◽

Dependent Manner ◽

Alizarin Red ◽

Sex Steroid Hormone ◽

Cos Cells ◽

Canine Osteosarcoma ◽

Multiple Parameters

Abstract While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019). Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and –secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

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Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

Communications Biology ◽

10.1038/s42003-020-01566-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Dasol Kim ◽

Hui-Yun Hwang ◽

Eun Sun Ji ◽

Jin Young Kim ◽

Jong Shin Yoo ◽

...

Keyword(s):

Metabolic Regulation ◽

Elongation Factor ◽

Dependent Manner ◽

Diet Induced Obesity ◽

Metabolic Dysregulation ◽

Autophagy Induction ◽

Transcription Factor Eb ◽

Mitochondrial Elongation

AbstractDisorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

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Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

Cell Death and Disease ◽

10.1038/s41419-021-03393-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wei Zhang ◽

Guoyu Yin ◽

Heping Zhao ◽

Hanzhi Ling ◽

Zhen Xie ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Hyaluronic Acid ◽

Dependent Manner ◽

Collagen Induced Arthritis ◽

Intracellular Degradation ◽

Main Pathway ◽

First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

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Phaseolin Attenuates Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells and Zebrafish

Biomedicines ◽

10.3390/biomedicines9040420 ◽

2021 ◽

Vol 9 (4) ◽

pp. 420

Author(s):

Su-Jung Hwang ◽

Ye-Seul Song ◽

Hyo-Jong Lee

Keyword(s):

Nitric Oxide ◽

Nuclear Translocation ◽

Raw 264.7 Cells ◽

Network Pharmacology ◽

Raw 264.7 ◽

Dependent Manner ◽

Bioactive Constituents

Kushen (Radix Sophorae flavescentis) is used to treat ulcerative colitis, tumors, and pruritus. Recently, phaseolin, formononetin, matrine, luteolin, and quercetin, through a network pharmacology approach, were tentatively identified as five bioactive constituents responsible for the anti-inflammatory effects of S. flavescentis. However, the role of phaseolin (one of the primary components of S. flavescentis) in the direct regulation of inflammation and inflammatory processes is not well known. In this study, the beneficial role of phaseolin against inflammation was explored in lipopolysaccharide (LPS)-induced inflammation models of RAW 264.7 macrophages and zebrafish larvae. Phaseolin inhibited LPS-mediated production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), without affecting cell viability. In addition, phaseolin suppressed pro-inflammatory mediators such as cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in a dose-dependent manner. Furthermore, phaseolin reduced matrix metalloproteinase (MMP) activity as well as macrophage adhesion in vitro and the recruitment of leukocytes in vivo by downregulating Ninjurin 1 (Ninj1), an adhesion molecule. Finally, phaseolin inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB). In view of the above, our results suggest that phaseolin could be a potential therapeutic candidate for the management of inflammation.

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PapRIV, a BV-2 microglial cell activating quorum sensing peptide

Scientific Reports ◽

10.1038/s41598-021-90030-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yorick Janssens ◽

Nathan Debunne ◽

Anton De Spiegeleer ◽

Evelien Wynendaele ◽

Marta Planas ◽

...

Keyword(s):

Quorum Sensing ◽

Cell Model ◽

Dependent Manner ◽

Communication Signals ◽

Gram Positive Bacteria ◽

A Cell ◽

Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

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Lumican Inhibits Osteoclastogenesis and Bone Resorption by Suppressing Akt Activity

International Journal of Molecular Sciences ◽

10.3390/ijms22094717 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4717

Author(s):

Jin-Young Lee ◽

Da-Ae Kim ◽

Eun-Young Kim ◽

Eun-Ju Chang ◽

So-Jeong Park ◽

...

Keyword(s):

Bone Resorption ◽

Bone Formation ◽

Map Kinases ◽

Osteoclast Differentiation ◽

Dependent Manner ◽

Dual Action ◽

Dose Dependent ◽

Resorptive Activity

Lumican, a ubiquitously expressed small leucine-rich proteoglycan, has been utilized in diverse biological functions. Recent experiments demonstrated that lumican stimulates preosteoblast viability and differentiation, leading to bone formation. To further understand the role of lumican in bone metabolism, we investigated its effects on osteoclast biology. Lumican inhibited both osteoclast differentiation and in vitro bone resorption in a dose-dependent manner. Consistent with this, lumican markedly decreased the expression of osteoclastogenesis markers. Moreover, the migration and fusion of preosteoclasts and the resorptive activity per osteoclast were significantly reduced in the presence of lumican, indicating that this protein affects most stages of osteoclastogenesis. Among RANKL-dependent pathways, lumican inhibited Akt but not MAP kinases such as JNK, p38, and ERK. Importantly, co-treatment with an Akt activator almost completely reversed the effect of lumican on osteoclast differentiation. Taken together, our findings revealed that lumican inhibits osteoclastogenesis by suppressing Akt activity. Thus, lumican plays an osteoprotective role by simultaneously increasing bone formation and decreasing bone resorption, suggesting that it represents a dual-action therapeutic target for osteoporosis.

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The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

Journal of Neurosurgery Spine ◽

10.3171/2020.10.spine201511 ◽

2021 ◽

pp. 1-5

Author(s):

Kari Hanson ◽

Carly Isder ◽

Kristen Shogren ◽

Anthony L. Mikula ◽

Lichun Lu ◽

...

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

High Dose ◽

Inhibitory Effects ◽

Alizarin Red ◽

Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

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Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00251.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. G814-G821 ◽

Cited By ~ 10

Author(s):

Bi-Guang Tuo ◽

Jimmy Y. C. Chow ◽

Kim E. Barrett ◽

Jon I. Isenberg

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Duodenal Mucosa ◽

Bicarbonate Secretion ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ussing Chambers ◽

Duodenal Bicarbonate Secretion

PKC has been shown to regulate epithelial Cl- secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current ( Isc). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or Isc ( P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (from 10-8 to 10-5M) ( P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCα, -γ, -ϵ, -θ, -μ, and -ι/λ were expressed in murine duodenal mucosa. Ro 31–8220 (an inhibitor active against PKCϵ, -α, -β, and -γ), but not Gö 6983 (an inhibitor active against PKCα, -γ, -β, and -δ), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCϵ, an effect that was prevented by Ro 31–8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCϵ isoform.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Preparation and In Vitro Characterization of Polycaprolactone and Demineralized Bone Matrix Scaffolds

MRS Proceedings ◽

10.1557/opl.2012.740 ◽

2012 ◽

Vol 1417 ◽

Author(s):

Titilayo Moloye ◽

Christopher Batich

Keyword(s):

Demineralized Bone Matrix ◽

Bone Matrix ◽

Apatite Formation ◽

Alizarin Red ◽

Salt Leaching ◽

Synthetic Materials ◽

Calcium And Phosphorus ◽

Demineralized Bone

ABSTRACTCylindrical porous polycaprolactone (PCL) scaffolds containing 25, 35, and 50 wt% demineralized bone matrix (DBM) were fabricated using a salt-leaching method for application in bone engineering. In the present work, PCL-DBM scaffolds were monitored for calcium and phosphorus deposition in both deionized (DI) water and simulated body fluid (SBF) for time periods of 5, 10, 15, and 20 days at 37°C under constant rotation. An in vitro assessment of the bioactivity of synthetic materials using SBF under physiological conditions can be used as a barometer of scaffold behavior in vivo. DBM, an osteoinductive material, was used to gauge if there was a correlation between the concentration of DBM within a scaffold and the apatite formation on its surface. Biochemical assays, alizarin red S staining, and scanning electron microscopy (SEM) with elemental analysis of calcium and phosphorus were consistent in that they confirmed that PCL scaffolds containing 35 wt% DBM in SBF at 14 days post-immersion showed signs of early apatite formation.

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