Jung Min Lim

Ca2+ Signaling Tools Acquired from Prostasomes Are Required for Progesterone-Induced Sperm Motility

Prostate-derived vesicles provide sperm with calcium signaling proteins required for progesterone-induced motility. Delivering Sperm a Toolkit Progesterone stimulates complex calcium signals in human sperm that have been implicated in enhancing their motility. Noting that sperm are structurally simple cells with a minimal complement of organelles, Park et al. explored the possibility that prostasomes (small vesicles secreted by the prostate) might deliver a “calcium toolkit” to sperm to enable these complex calcium responses. Analyses of sperm isolated before prostasome exposure revealed that, indeed, fusion with prostasomes led to the acquisition in sperm of various proteins implicated in calcium signaling and enabled the progesterone-dependent mobilization of calcium from internal stores. Moreover, prostasomal proteins promoted progesterone-dependent sperm motility and enhanced the ability of mouse sperm to fertilize ova. Progesterone-induced calcium ion (Ca2+) signals in the neck region of sperm play a pivotal role in promoting sperm motility. Here, we show that a long-lasting Ca2+ signal required for sperm motility in response to progesterone depends on their pH-dependent fusion with prostasomes, which are small vesicles secreted by the prostate. We found that prostasome fusion led to the transfer of progesterone receptors, cyclic adenosine diphosphoribose (cADPR)–synthesizing enzymes, ryanodine receptors (RyRs), and other Ca2+ signaling tools from prostasomes to the sperm neck. Progesterone-induced sperm motility relied on cADPR-mediated Ca2+ mobilization through RyR located on acidic Ca2+ stores, followed by Ca2+ entry through store-operated channels. Treatment of prostasome-fused sperm with a cADPR antagonist or fusion with prostasomes in which type 2 RyR was depleted resulted in low fertilization rates, reduced sperm motility, or both. Thus, we conclude that sperm motility depends on the acquisition of Ca2+ signaling tools from prostasomes.

Angiotensin-(1-7) attenuates hypertension in exercise-trained renal hypertensive rats.

Angiotensin-(1-7) [ANG-(1-7)] plays a counterregulatory role to angiotensin II in the renin-angiotensin system. In trained spontaneous hypertensive rats, Mas expression and protein are upregulated in ventricular tissue. Therefore, we examined the role of ANG-(1-7) on cardiac hemodynamics, cardiac functions, and cardiac remodeling in trained two-kidney one-clip hypertensive (2K1C) rats. For this purpose, rats were divided into sedentary and trained groups. Each group consists of sham and 2K1C rats with and without ANG-(1-7) infusion. Swimming training was performed for 1 h/day, 5 days/wk for 4 wk following 1 wk of swimming training for acclimatization. 2K1C rats showed moderate hypertension and left ventricular hypertrophy without changing left ventricular function. Chronic infusion of ANG-(1-7) attenuated hypertension and cardiac hypertrophy only in trained 2K1C rats but not in sedentary 2K1C rats. Chronic ANG-(1-7) treatment significantly attenuated increases in myocyte diameter and cardiac fibrosis induced by hypertension in only trained 2K1C rats. The Mas receptor, ANG II type 2 receptor protein, and endothelial nitric oxide synthase phosphorylation in ventricles were upregulated in trained 2K1C rats. In conclusion, chronic infusion of ANG-(1-7) attenuates hypertension in trained 2K1C rats.

Enhanced Lysosomal Activity Is Involved in Bax Inhibitor-1-induced Regulation of the Endoplasmic Reticulum (ER) Stress Response and Cell Death against ER Stress INVOLVEMENT OF VACUOLAR H+-ATPASE (V-ATPASE)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H+-ATPase(V-ATPase), a proton pump, was activated, suggesting high H+ uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.

Enhancement of tibial regeneration in a rat model by adipose-derived stromal cells in a PLGA scaffold

INTRODUCTION Autologous adipose-derived stromal cells (ASCs) are an obvious source of osteogenic cells and can be easily isolated from adipose tissue. We evaluated the potential of ASCs seeded onto a scaffold to heal tibial defects. METHODS Autologous ASCs were obtained from adipose tissue by collagenase digestion. The cells were seeded in three-dimensional poly(lactic)-glycolic acid (PLGA) scaffolds and cultured in osteogenic medium for four weeks. Evidence of osteogenesis was assessed by von Kossa staining in three-dimensional cultures following osteogenic induction. The critical size tibial defects (10mm) were created using a rat model. Defects were either left empty (sham group), treated with a PLGA scaffold alone (PLGA group), or a PLGA/ASC composite (PLGA/ASC group). Using radiologic and histologic analyses, we assessed total bone volume and vascular density. Total RNA was prepared from regenerated bone and analyzed for osteogenic marker gene expression. RESULTS In three-dimensional cultures, the PLGA/ASC composite showed multiple calcified extracellular matrix nodules on von Kossa staining after four weeks of differentiation. Near complete healing was observed between the PLGA/ASC engrafted tibial defects on plain radiographs and micro-CT findings. Total bone volume and mechanical strength were significantly higher in the PLGA/ASC group compared to the sham and PLGA groups. Histologic analysis revealed increased new bone formation along capillaries in the PLGA/ASC group. Real-time RT-PCR analysis revealed a significant increase in the expression of osteogenic genes in the PLGA/ASC group. CONCLUSIONS The results showed that the repair of tibial defects was accelerated by implantation of autologous ASCs seeded onto a PLGA scaffold. Therefore, PLGA/ASC is a promising new cell-based therapy for healing critical size tibial defects.

n-3 Polyunsaturated fatty acids protect against pancreatic β-cell damage due to ER stress and prevent diabetes development

SCOPE In this study, we focus on the effects of n-3 polyunsaturated fatty acids (PUFAs) on tunicamycin-, streptozotocin-, or high fat diet (HFD)-induced β-cell damage and dysfunction. MATERIALS AND METHODS Pretreatment with n-3 PUFAs protected RINm5F cells and mouse islets against tunicamycin-induced β-cell damage through suppression of ER stress and apoptosis induction. This protective effect of n-3 PUFAs on β-cells was further demonstrated by the normalization of insulin secretion in response to glucose in tunicamycin-treated islets. In multiple low-dose streptozotocin-induced diabetes models, fat-1 mice, which endogenously synthesize n-3 PUFAs from n-6 PUFAs, were fully resistant to the development of diabetes, with normal islet morphology, high insulin immunoreactivity, and decreased apoptotic cells. In HFD-induced diabetes models, fat-1 mice also exhibited improved glucose tolerance and functional β-cell mass. In both diabetes models, we observed an attenuation of ER stress in fat-1 mice. Interestingly, n-3 PUFAs attenuated the nuclear translocation of lipogenic transcription factors sterol regulatory element-binding protein-1 (SREBP-1) and C/EBPβ, induced by tunicamycin or HFD, suggesting that n-3 PUFAs suppress ER stress via modulation of SREBP-1 and C/EBPβ. CONCLUSION Together, these results suggest that n-3 PUFAs block ER stress, thus protecting β cells against diabetogenic insult; therefore, dietary supplementation of n-3 PUFAs has therapeutic potential for the preservation of functional β-cell mass.

Polyphenols isolated from Broussonetia kazinoki prevent cytokine-induced β-cell damage and the development of type 1 diabetes

The axis of nuclear factor κB (NF-κB)-inducible NO synthase (iNOS)-nitric oxide plays a key role in cytokine- and streptozotocin-mediated pancreatic β-cell damage. In this study, we investigated the effects of kazinol C and isokazinol D isolated from Broussonetia kazinoki on the β-cell viability and function. RINm5F cells and primary islets were used for in vitro and ex vivo cytokine toxicity experiments, respectively. For type 1 diabetes induction, mice were injected with multiple low-dose streptozotocin (MLDS). Cytokine-induced toxicity was completely abolished in both RINm5F cells and islets that were pretreated with either kazinol C or isokazinol D. Both kazinols inhibited the NF-κB signaling pathway, thereby inhibiting cytokine-mediated iNOS induction, nitric oxide production, apoptotic cell death and defects in insulin secretion. Moreover, the occurrence of diabetes in MLDS-treated mice was efficiently attenuated in kazinol-pretreated mice. Immunohistochemical analysis revealed that the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cells and nuclear p65-positive cells were significantly decreased in kazinol-pretreated mice. Our results suggest that kazinol C and isokazinol D block the NF-κB pathway, thus reducing the extent of β-cell damage. Therefore, kazinol C and isokazinol D may have therapeutic value in delaying pancreatic β-cell damage in type 1 diabetes.

Caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion

Caveolae may act as mechanosensors and function as binding sites for calcium ions. The intracaveolar localization of atrial natriuretic peptide (ANP) derived from the direct interaction of atrial granules with caveolae has been demonstrated. The aim of this study was to define the effect of caveolae on ANP secretion induced by stretch and angiotensin II. The isolated perfused beating atria from Sprague-Dawley rats were used. To disrupt caveolae, 10mM methyl-β-cyclodextrin (MbCD) was applied for 1h and the number of caveoli were markedly decreased. MbCD increased basal ANP secretion and atrial diastolic pressure. The molecular profile of ANP in perfusate from control atria showed mainly one major peak corresponded to synthetic ANP whereas that from MbCD-treated atria showed two major immunoreactive peaks corresponded to synthetic rat ANP and proANP. High atrial stretch induced by elevating the height of outflow catheter from 5 cm H₂O to 7.5 cm H₂O increased atrial contractility and ANP secretion. The response of ANP secretion to high stretch was attenuated in MbCD-pretreated atria. Pretreatment with MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. However, the effect of angiotenisin (1-7) on ANP secretion was not altered by MbCD treatment. The expression of angiotensin II type 1 receptor protein was reduced by MbCD treatment. These data suggest that caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion and relate to the processing of proANP.

SPA0355 suppresses T-cell responses and reduces airway inflammation in mice.

In recent studies, SPA0355, a thiourea analog, has been demonstrated to possess strong anti-inflammatory activity. However, the mechanisms underlying the effects of SPA0355 on immune-mediated diseases have not been fully defined. The present study was designed to investigate the immunological and molecular mechanisms by which SPA0355 modulates cluster of differentiation of (CD4)(+) T-cell-mediated immune responses in allergic airway inflammation. In vitro studies have shown that SPA0355 suppresses CD4(+) T-cell activation, proliferation, and differentiation via modulation of T-cell receptor (TCR) signal transduction and cytokine-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Next, we investigated the efficacy of SPA0355 in ovalbumin (OVA)-induced allergic airway inflammation. Intraperitoneal administration of SPA0355 inhibited inflammatory cell recruitment into the airways as well as the production of Th2 cytokines in bronchoalveolar fluid and suppressed OVA-induced IgE production in serum. Additionally, SPA0355 suppressed mucin production and smooth muscle hypertrophy and prevented the development of airway hyperresponsiveness. Given that allergic airway inflammation is mainly driven by Th2 cell responses, it is highly possible that the defects in CD4(+) T-cell activation and Th2 cell differentiation in the draining lymph nodes and suppressed NF-κB activation in the lungs of SPA0355-treated mice illustrate an immunological mechanism of the preventive effect of SPA0355 on the aforementioned asthmatic characteristics. Collectively, our results suggest that SPA0355 directly modulates Th1 and Th2 responses through the suppression of multiple signaling pathways triggered by TCR or cytokine receptor stimulation, and that SPA0355 has protective effects in a murine model of allergic airway inflammation.