Absence Of Y-27632 Research Articles

Y-27632 enables long-term expansion of mouse submandibular gland epithelial cells via inactivation of TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathway

ObjectivesThis study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms. MethodsThe role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632. ResultsTreatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 indicated that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance. ConclusionsThe TGF-β1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

ROCK inhibition modulates the senescence-associated secretory phenotype (SASP) in oral keratinocytes.

Senescent cells accumulate in different organs and develop a senescence-associated secretory phenotype (SASP), associated with the development of age-related pathologies. The constitution of the SASP varies among cell types and with the method of senescence induction; nevertheless, there is substantial overlap among SASPs, especially the presence of pro-inflammatory cytokines such as IL-1β, IL-1α, IL-6 and IL-8. These cytokines are highly conserved among SASPs and are implicated in the development of several cancers. Here, we report that ROCK inhibition by Y-27632 reduces levels of IL-1α, IL-1β, IL-6 and IL-8 secreted by senescent normal and dysplastic oral keratinocytes without affecting the permanent cell growth arrest. The data indicate some inflammatory genes downregulated by Y-27632 remain downregulated even after repeated passage in the absence of Y-27632. We propose ROCK kinase inhibition as a novel alternative to current strategies to modulate the inflammatory components of the SASP, without compromising the permanent cell growth arrest. This observation potentially has wide clinical applications, given the involvement of senescence in cancer and a wide range of age-related disease. It also suggests care should be exercised when using Y-27632 to facilitate cell expansion of primary cells, as its effects on gene expression are not entirely reversible.

Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.

Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10μM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.

Effects of Y-27632, a rho-associated kinase ROCK inhibitor, on adhesion, survival, syncytium formation and hCG secretion of term human cytotrophoblasts

s / Placenta 35 (2014) A1eA112 A101 P2.128-N. EFFECTS OF Y-27632, A RHO-ASSOCIATED KINASE ROCK INHIBITOR, ON ADHESION, SURVIVAL, SYNCYTIUM FORMATION AND HCG SECRETION OF TERM HUMAN CYTOTROPHOBLASTS Kenichiro Motomura , Naoko Okada , Akio Matsuda , Haruhiko Sago , Hirohisa Saito , Kenji Matsumoto a National research institute for child health and development, Tokyo, Japan; National center for child health and development, Tokyo, Japan Objectives: Several different signal transduction pathways are known to participate in the multiple steps of placenta formation. Rho kinase reportedly promotes first-trimester extravillous trophoblast migration, but the effects of Rho-associated kinase on the cytotrophoblasts themselves remain obscure. In the present study, we investigated the effects of Y27632, a Rho-associated kinase ROCK inhibitor, on several functions of term human cytotrophoblasts. Methods: Primary human trophoblasts (PHT) were isolated from term uncomplicated placentas by trypsin, dispase and DNase I treatment, percoll density gradient centrifugation and negative selection using anti-HLA class I mAb-coated immunomagnetic beads. Purified PHT were suspended in 10% FBS-containing medium supplemented with EGF in the presence and absence of Y-27632 and cultured in non-coated plates for up to 96 hours. Cell viability was measured by WST-8 assay. Adhesion was assessed by counting the number of cell nuclei on the culture plate after staining with an immunofluorescent dye. Syncytium formation and trophoblast maturation were assessed by qPCR assay of syncytin1/syncytin2 and betahCG mRNA expression, respectively. Results: Y-27632 significantly increased PHT viability. Y-27632 increased PHT adhesion to the non-coated culture plate in a concentration-dependent manner. Y-27632 enhanced expression of mRNA for such syncytialization markers as syncytin1 and syncytin2. Y-27632 also enhanced expression of beta-hCG mRNA. Conclusion: Rho-associated kinase is likely to play critical roles in several functions of PHT. The mechanisms regulating Rho-associated kinase in cytotrophoblasts should be studied further. In addition, intracellular signals downstream of Rho-associated kinase should be clarified. P2.129-N. TO INVADE OR NOT TO INVADE: DEFINING THE ROLE OF MTA3 IN TROPHOBLAST DIFFERENTIATION AND MIGRATION Mariko Horii, Matteo Moretto-Zita, Yingchun Li, Mana M. Parast Department of Pathology, University of California San Diego, La Jolla,

The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional.

IntroductionWe previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.MethodsIn this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.ResultsWe demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.ConclusionsROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.

The role of Rho-kinases in IL-1β release through phagocytosis of fibrous particles in human monocytes.

Long fibers, such as asbestos and carbon nanotubes (CNTs), are more potent activators of inflammatory and genotoxicity than short or tangled fibers. Fibrous particles trigger interleukin (IL)-1β secretion and cause inflammatory diseases through NLRP3 inflammasomes in phagocytotic cells. However, the mechanism involved in fibrous particle-induced inflammation has not been well documented. In this study, we focused on GTPase effector Rho-kinases (ROCK1, and 2), which are known to be involved in a wide range of cellular functions such as adhesion, regulation of cytoskeleton, and phagocytosis. We examined whether ROCKs are associated with multi-walled CNT (MWCNT)- or asbestos-induced IL-1β secretion in human monocytic THP-1 cells using a selective inhibitor and small interfering RNA. THP-1 cells were differentiated to macrophages by PMA and were exposed to MWCNTs, crocidolite asbestos or lipopolysaccharide (LPS) in the presence or absence of Y27632 (ROCK inhibitor) or Z-YVAD (caspase-1 inhibitor). Exposure of the cells to MWCNTs or asbestos provoked IL-1β secretion, but this secretion was suppressed by both Y27632 and Z-YVAD, whereas LPS-induced IL-1β secretion was inhibited only by Z-YVAD and not by Y27632. siRNA designed for knockdown of both ROCK1 and ROCK2 suppressed MWCNT- and asbestos-induced IL-1β secretion, but did not change LPS-induced IL-1β secretion. Moreover, Y27632 suppressed pro-IL-1β protein levels and the release of activated-cathepsin B and activated-caspase-1 induced by MWCNTs or asbestos. In contrast, LPS-induced pro-IL-1β protein was not suppressed by Y27632. These results suggest that ROCKs are involved in fibrous particle-induced inflammasome responses in THP-1 cells.

Mepivacaine-induced contraction involves increased calcium sensitization mediated via Rho kinase and protein kinase C in endothelium-denuded rat aorta

Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca2+]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration–response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca2+]i-tension curve was higher than that of the KCl-induced [Ca2+]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca2+]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways.

Abstract 494: Rho-a Kinase Activation in Endothelial Cells is Induced by Cocaine or Plasma From Cocaine Consumers: Association With a Prothrombotic Phenotype and Effect of the Inhibitors Atorvastatin and Y-27632

Cocaine consumption is associated with increased risk of cardio- and cerebrovascular diseases, which are the consequence of endothelial dysfunction (ED). We recently showed that cocaine use is related to increased ED markers including circulating endothelial cells, although the mechanisms governing ED in cocaine users are still being researched. Since the RhoA/Rho kinase (ROCK) pathway is involved in the pathogenesis of ED, our Aim was to study in vitro and ex vivo RhoA/Rho kinase activation and ED markers in relation to cocaine consumption. Method Plasma samples from active cocaine consumers who met DSM-IV criteria and age and gender-matched controls were tested. ROCK activity, von Willebrand factor (vWf), Metalloproteases (MMP) and platelet adhesion were evaluated in HUVEC supplemented with cocaine (10μM), 10% plasma from cocaine users (CP), normal plasma (NP) or vehicle, in the presence or absence of Y-27632 (10 μM) and atorvastatin (ATS:10μM). Results CP induced higher ROCK (p: 0.039) and MMP 2 activity (p<0.05) in HUVEC, released significantly elevated vWf (p0.001), but failed to stimulate MMP activity or vWf release. ATS and Y-27632 significantly reduced ROCK activity (p<0.01 and p<0.001) and platelet adhesion (p< 0.0001 and p: 0.0073) in CP treated cells. ATS also reduced MMP2 activity (p< 0.001) and vWf release (p: 0.004). In cocaine treated cells, ATS and Y-27632 downregulated ROCK activity (p: 0.039 and p: 0.091) and significantly reduced platelet adhesion (p: 0.0156 and p: 0.073). Ex vivo studies showed significantly higher ROCK (p 0.015), MMP-2 (p<0.05) and MMP-9 (p<0.01) activity in cocaine consumers than in controls. Conclusions Cocaine and cocaine consumer plasma increased ROCK activity in HUVEC, resembling the ex vivo findings. Only CP induced higher MMP activity and release of vWf resembling the findings in patients. The significant reduction of most of the in vitro effects by ATS and Y-27632 may provide a novel and potential therapeutic tool for the protection of the endothelium from deleterious stimuli such as cocaine consumption.

Nocodazole Treatment Decreases Expression of Pluripotency Markers Nanog and Oct4 in Human Embryonic Stem Cells

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition, the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle, which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4.

ROCK1/2 Inhibitor Induces Enteric Phenotype in CNS Derived Neural Stem Cells

Introduction Central nervous system-derived neural stem cells (CNS-NSC) have been previously shown to populate the enteric nervous system after transplantation in the gut. CNSNSC, are therefore considered promising candidates for cell based therapy for enteric neuropathies such as achalasia and Hirschsprung's disease. However the optimal conditions leading to the most efficient conversion of CNS-NSC to an enteric phenotype are still being investigated. Here we describe a simple and efficient method of inducing enteric phenotype in CNSNSC using the ROCK1/2 inhibitor Y27632. Methods CNS-NSC isolated from the subventricular zone of E13.5 mouse embryos were cultured under proliferating conditions in presence or absence of 25uM Y27632 and the expression of enteric markers was examined by qRT-PCR after 5 days. Results The CNS-NSC cultured in presence of Y27632 formed neurospheres that adhered and cells started migrating out of them and growing neural processes. On the other hand, CNS-NSC cultured in the absence of Y27632 continued to proliferate and grow as floating neurospheres. qRT-PCR analysis of cells cultured with Y27632 revealed 2.5-fold increase in the expression of p75, an enteric neural progenitor marker and 4.5 fold increase in the expression of Ret, a key regulator of enteric neuronal development over cells cultured without Y27632. The expression of Sox 10, a glial cell marker, was however found to be 3.3 fold lower in cells cultured with Y27632, suggesting preferential differentiation of CNS-NSC into neurons in presence of Y27632. Conclusions Inhibition of ROCK1/2 is a novel, highly efficient and less manipulative strategy for inducing enteric phenotype in NSC derived from CNS and it can potentially be expanded to induce enteric phenotype on stem cells of diverse origin.

Effect of the Rho kinase inhibitor Y‐27632 on the proteome of hearts with ischemia–reperfusion injury

Growing attention has been given to the role of the Rho kinase pathway in the development of heart disease and ischemia/reperfusion (I/R) injury. Y-27632 is a Rho kinase inhibitor demonstrated to protect against I/R injury, but the exact mechanism by which it does so remains to be elucidated. The goal of this project was to determine new targets by which Y-27632 can protect the heart against I/R injury. Isolated rat hearts were perfused under aerobic conditions or subjected to I/R in the presence or absence of Y-27632. Administration of Y-27632 (1 μM) before ischemia and during the first 10 min of reperfusion resulted in complete recovery of cardiac function. 2-D electrophoresis followed by MS identified four proteins whose levels were affected by Y-27632 treatment. Lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were significantly increased in the Y-27632 treated group, while creatine kinase was normalized to control levels. In addition, we found increased level of two different molecular fragments of ATP synthase, which were normalized by Y-27632. This increase suggests that during ischemia ATP synthase is subjected to degradation. The changes in metabolic enzymes' levels and their regulation by Y-27632 suggest that the cardioprotective effect of Y-27632 involves increased energy production.

The ROCK Inhibitor Y-27632 Improves Recovery of Human Embryonic Stem Cells after Fluorescence-Activated Cell Sorting with Multiple Cell Surface Markers

BackgroundDue to the inherent sensitivity of human embryonic stem cells (hESCs) to manipulations, the recovery and survival of hESCs after fluorescence-activated cell sorting (FACS) can be low. Additionally, a well characterized and robust methodology for performing FACS on hESCs using multiple-cell surface markers has not been described. The p160-Rho-associated coiled kinase (ROCK) inhibitor, Y-27632, previously has been identified as enhancing survival of hESCs upon single-cell dissociation, as well as enhancing recovery from cryopreservation. Here we examined the application of Y-27632 to hESCs after FACS to improve survival in both feeder-dependent and feeder-independent growth conditions.Methodology/Principal FindingsHESCs were sorted using markers for SSEA-3, TRA-1-81, and SSEA-1. Cells were plated after sorting for 24 hours in either the presence or the absence of Y-27632. In both feeder-dependent and feeder-independent conditions, cell survival was greater when Y-27632 was applied to the hESCs after sort. Specifically, treatment of cells with Y-27632 improved post-sort recovery up to four fold. To determine the long-term effects of sorting with and without the application of Y-27632, hESCs were further analyzed. Specifically, hESCs sorted with and without the addition of Y-27632 retained normal morphology, expressed hESC-specific markers as measured by immunocytochemistry and flow cytometry, and maintained a stable karyotype. In addition, the hESCs could differentiate into three germ layers in vitro and in vivo in both feeder-dependent and feeder-independent growth conditions.Conclusions/SignificanceThe application of Y-27632 to hESCs after cell sorting improves cell recovery with no observed effect on pluripotency, and enables the consistent recovery of hESCs by FACS using multiple surface markers. This improved methodology for cell sorting of hESCs will aid many applications such as removal of hESCs from secondary cell types, identification and isolation of stem cell subpopulations, and generation of single cell clones. Finally, these results demonstrate an additional application of ROCK inhibition to hESC research.

RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC)

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.

Rho kinase inhibitor Y-27632 facilitates recovery from experimental peripheral neuropathy induced by anti-cancer drug cisplatin

Chemotherapy drugs have neurotoxicity associated with treatment, which can become a dose-limiting problem when clinical presentation is severe. However, there is no effective therapy to circumvent the neurotoxicity of anti-cancer drug treatment. In this study, we utilized a newly designed mouse model of cisplatin-induced peripheral neuropathy to determine both the severity of neurotoxicity induced by drug treatment and the effectiveness of the Rho kinase inhibitor Y-27632 in post-treatment recovery. Sensory nerve conduction studies revealed a significant increase in mean distal (peak) latency with cisplatin treatment, indicating a deterioration of sensory nerve function. Also, hind paw touch sensitivity decreased steadily with increasing cumulative dose of cisplatin. Histological and immunohistochemical analyses of the sural nerve using neuronal marker protein gene product 9.5 (PGP 9.5) demonstrated abnormal nerve fiber morphology in cisplatin-treated mice. Remarkably, post-treatment with Y-27632 improved the sural nerve distal (peak) latency and sensory threshold to return to pre-treatment levels. Sural nerve histology worsened in the absence of Y-27632 during recovery. These studies suggest that Rho kinase inhibitor Y-27632 can initiate regeneration of damaged nerves following cisplatin treatment.

Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm

Human embryonic stem cells (hESCs) are potential renewable sources of cells in replacement therapies for many diseases including type 1 diabetes. We have established a three dimensional (3D) model to culture and differentiate hESCs that are encapsulated in calcium alginate microcapsules. This system promotes cellular interactions that are essential for both maintaining pluripotency and differentiation. This 3D model also provides opportunity to separate out hESCs from fibroblasts used as feeder layer during culture. In this study, we compared the viability and proliferation of the encapsulated hESCs cultured in serum replacement (SR) medium, human fetal fibroblast-conditioned medium (hFF-CM), in the presence and absence of Y-27632, a ROCK inhibitor. Treatment of hESCs with Y-27632 promoted cell survival, cell cluster formation and proliferation rate in both SR medium and hFF-CM. These encapsulated hESC clusters were then directly differentiated to definitive endoderm cells that expressed mesendoderm (Brachyury 70-fold), definitive endoderm (SOX17 > 300-fold, FOXA2 > 800-fold, and CXCR4 > 100-fold) and primitive gut tube (HNF1β > 120-fold) as compared to the undifferentiated hESCs. These data show that microcapsules can be used for differentiation of hESCs into definitive endoderm in 3D and could have potential application for immune-isolation and prevention of teratomas formation of hESCs during transplantation.

Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2-4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor

The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs. MCECs of cynomolgus monkeys were cultured in a medium containing 10 microM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Prostate-derived Sterile 20-like Kinase 2 (PSK2) Regulates Apoptotic Morphology via C-Jun N-terminal Kinase and Rho Kinase-1

We have reported previously that human prostate-derived sterile 20-like kinase (PSK) 1 alters actin cytoskeletal organization and binds to microtubules, regulating their organization and stability. We have shown a structurally related protein kinase PSK2, which lacks a microtubule-binding site, activated c-Jun N-terminal kinase (JNK), and induced apoptotic morphological changes that include cell contraction, membrane blebbing, and apoptotic body formation. Apoptotic stimuli increased the catalytic activity of endogenous PSK2 and JNK, and dominant negative JNK or a physiological inhibitor of JNK blocked these apoptotic morphological responses to PSK2, demonstrating a requirement for JNK. PSK2 also stimulated the cleavage of Rho kinase-1 (ROCK-I), and the activity of ROCK-I was required for PSK2 to induce cell contraction and membrane blebbing. The activation of caspases was also needed for the induction of membrane blebbing by PSK2, which was itself a substrate for caspase 3. PSK2 therefore regulates apoptotic morphology associated with the execution phase of apoptosis, which involves dynamic reorganization of the actin cytoskeleton, via downstream targets that include JNK and ROCK-I. Our findings suggest that PSKs form a subgroup of sterile 20 (STE20)-like kinases that regulate different cytoskeletal processes.

Independence of plasma membrane blebbing from other biochemical and biological characteristics of apoptotic cells.

Plasma membrane blebs are observed in many types of apoptotic cells, but their physiological roles remain to be clarified. We examined whether there is a causative connection between membrane blebbing and other apoptotic changes in Jurkat cells induced to undergo apoptosis by doxorubicin in the presence or absence of Y-27632, an inhibitor of the Rho kinase ROCK-I. The inclusion of the drug made most membrane blebs disappear, while other changes, such as chromatin condensation, inactivation of mitochondrial enzymes, externalization of the membrane phospholipid phosphatidylserine, and removal of cell surface sialic acid, remained unaffected. Furthermore, these apoptotic cells were phagocytosed by macrophages as efficiently as normally apoptosing cells. These results indicate that blebbing of the plasma membrane occurs independently from other apoptotic changes and is not involved in the recognition and engulfment of apoptotic cells by macrophages.