The Rho-Rock-Myosin signaling axis determines cell-cell integrity of self-renewing pluripotent stem cells

Background

Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity, molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.

Methodology/Principal Findings

Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock, a major effector kinase downstream of Rho, played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore, a novel culture system combining a single synthetic matrix, defined medium, and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells as determined by teratoma formation assay.

Conclusions/Significance

These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells, and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells.     

Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide

Background

N-butylidenephthalide (BP) exhibits antitumor effect in a variety of cancer cell lines. The objective of this study was to obtain additional insights into the mechanisms involved in BP induced cell death in human prostate cancer cells.

Methods/Principal Findings

Two human prostate cancer cell lines, PC-3 and LNCaP, were treated with BP, and subsequently evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest and cell death in both cell lines. The G0/G1 phase arrest was correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. To determine the mechanisms of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by BP in the LNCaP cells. Several BP-induced genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. BP-induced ER stress was evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-α and GADD153/CHOP in both cell lines. Blockage of IRE1-α or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Furthermore, blockage of JNK1/2 signaling by JNK siRNA resulted in decreased expression of IRE1-α and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. BP also suppressed LNCaP xenograft tumor growth in NOD-SCID mice. It caused 68% reduction in tumor volume after 18 days of treatment.

Conclusions

Our results suggest that BP can cause G0/G1 phase arrest in prostate cancer cells and its cytotoxicity is mediated by ER stress induction. Thus, BP may serve as an anticancer agent by inducing ER stress in prostate cancer.     

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background

Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics.

Methods and Findings

To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF.

Conclusions

These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.     

STI571 reduces TRAIL-induced apoptosis in colon cancer cells: c-Abl activation by the death receptor leads to stress kinase-dependent cell death

Background

In an effort to achieve better cancer therapies, we elucidated the combination cancer therapy of STI571 (an inhibitor of Bcr-Abl and clinically used for chronic myelogenous leukemia) and TNF-related apoptosis-inducing ligand (TRAIL, a developing antitumor agent) in leukemia, colon, and prostate cancer cells.

Methods

Colon cancer (HCT116, SW480), prostate cancer (PC3, LNCaP) and leukemia (K562) cells were treated with STI571 and TRAIL. Cell viability was determined by MTT assay and sub-G1 appearance. Protein expression and kinase phosphorylation were determined by Western blotting. c-Abl and p73 activities were inhibited by target-specific small interfering (si)RNA. In vitro kinase assay of c-Abl was conducted using CRK as a substrate.

Results

We found that STI571 exerts opposite effects on the antitumor activity of TRAIL. It enhanced cytotoxicity in TRAIL-treated K562 leukemia cells and reduced TRAIL-induced apoptosis in HCT116 and SW480 colon cancer cells, while having no effect on PC3 and LNCaP cells. In colon and prostate cancer cells, TRAIL caused c-Abl cleavage to the active form via a caspase pathway. Interestingly, JNK and p38 MAPK inhibitors effectively blocked TRAIL-induced toxicity in the colon, but not in prostate cancer cells. Next, we found that STI571 could attenuate TRAIL-induced c-Abl, JNK and p38 activation in HCT116 cells. In addition, siRNA targeting knockdown of c-Abl and p73 also reduced TRAIL-induced cytotoxicity, rendering HCT116 cells less responsive to stress kinase activation, and masking the cytoprotective effect of STI571.

Conclusions

All together we demonstrate a novel mediator role of p73 in activating the stress kinases p38 and JNK in the classical apoptotic pathway of TRAIL. TRAIL via caspase-dependent action can sequentially activate c-Abl, p73, and stress kinases, which contribute to apoptosis in colon cancer cells. Through the inhibition of c-Abl-mediated apoptotic p73 signaling, STI571 reduces the antitumor activity of TRAIL in colon cancer cells. Our results raise additional concerns when developing combination cancer therapy with TRAIL and STI571 in the future.     

Adult Cardiac Progenitor Cell Aggregates Exhibit Survival Benefit Both In Vitro and In Vivo

Background

A major hurdle in the use of exogenous stems cells for therapeutic regeneration of injured myocardium remains the poor survival of implanted cells. To date, the delivery of stem cells into myocardium has largely focused on implantation of cell suspensions.

Methodology and Principal Findings

We hypothesize that delivering progenitor cells in an aggregate form would serve to mimic the endogenous state with proper cell-cell contact, and may aid the survival of implanted cells. Microwell methodologies allow for the culture of homogenous 3D cell aggregates, thereby allowing cell-cell contact. In this study, we find that the culture of cardiac progenitor cells in a 3D cell aggregate augments cell survival and protects against cellular toxins and stressors, including hydrogen peroxide and anoxia/reoxygenation induced cell death. Moreover, using a murine model of cardiac ischemia-reperfusion injury, we find that delivery of cardiac progenitor cells in the form of 3D aggregates improved in vivo survival of implanted cells.

Conclusion

Collectively, our data support the notion that growth in 3D cellular systems and maintenance of cell-cell contact improves exogenous cell survival following delivery into myocardium. These approaches may serve as a strategy to improve cardiovascular cell-based therapies.     

Misregulated E-cadherin expression associated with an aggressive brain tumor phenotype

Background

Cadherins are essential components of the adherens junction complexes that mediate cell-cell adhesion and regulate cell motility. During tissue morphogenesis, changes in cadherin expression (known as cadherin switching) are a common mechanism for altering cell fate. Cadherin switching is also common during epithelial tumor progression, where it is thought to promote tumor invasion and metastasis. E-cadherin is the predominant cadherin expressed in epithelial tissues, but its expression is very limited in normal brain.

Methodology/Principal Findings

We identified E-cadherin expression in a retrospective series of glioblastomas exhibiting epithelial or pseudoepithelial differentiation. Unlike in epithelial tissues, E-cadherin expression in gliomas correlated with an unfavorable clinical outcome. Western blotting of two panels of human GBM cell lines propagated either as xenografts in nude mice or grown under conventional cell culture conditions confirmed that E-cadherin expression is rare. However, a small number of xenograft lines did express E-cadherin, its expression correlating with increased invasiveness when the cells were implanted orthotopically in mouse brain. In the conventionally cultured SF767 glioma cell line, E-cadherin expression was localized throughout the plasma membrane rather than being restricted to areas of cell-cell contact. ShRNA knockdown of E-cadherin in these cells resulted in decreased proliferation and migration in vitro.

Conclusions/Significance

Our data shows an unexpected correlation between the abnormal expression of E-cadherin in a subset of GBM tumor cells and the growth and migration of this aggressive brain tumor subtype.     

Angiotensin II Upregulates Endothelial Lipase Expression via the NF-Kappa B and MAPK Signaling Pathways

Background

Angiotensin II (AngII) participates in endothelial damage and inflammation, and accelerates atherosclerosis. Endothelial lipase (EL) is involved in the metabolism and clearance of high density lipoproteins (HDL), the serum levels of which correlate negatively with the onset of cardiovascular diseases including atherosclerosis. However, the relationship between AngII and EL is not yet fully understood. In this study, we investigated the effects of AngII on the expression of EL and the signaling pathways that mediate its effects in human umbilical vein endothelial cells (HUVECs).

Methods and Findings

HUVECs were cultured in vitro with different treatments as follows: 1) The control group without any treatment; 2) AngII treatment for 0 h, 4 h, 8 h, 12 h and 24 h; 3) NF-κB activation inhibitor pyrrolidine dithiocarbamate (PDTC) pretreatment for 1 h before AngII treatment; and 4) mitogen-activated protein kinase (MAPK) p38 inhibitor (SB203580) pretreatment for 1 h before AngII treatment. EL levels in each group were detected by immunocytochemical staining and western blotting. HUVECs proliferation was detected by MTT and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. NF-kappa B (NF-κB) p65, MAPK p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and phosphorylated extracellular signal-regulated kinase (p-ERK) expression levels were assayed by western blotting. The results showed that the protein levels of EL, NF-κB p65, MAPK p38, JNK, and p-ERK protein levels, in addition to the proliferation of HUVECs, were increased by AngII. Both the NF-kB inhibitor (PDTC) and the MAPK p38 inhibitor (SB203580) partially inhibited the effects of AngII on EL expression.

Conclusion

AngII may upregulate EL protein expression via the NF-κB and MAPK signaling pathways.     

LRP-1 Promotes Cancer Cell Invasion by Supporting ERK and Inhibiting JNK Signaling Pathways

Background

The low-density lipoprotein receptor-related protein-1 (LRP-1) is an endocytic receptor mediating the clearance of various extracellular molecules involved in the dissemination of cancer cells. LRP-1 thus appeared as an attractive receptor for targeting the invasive behavior of malignant cells. However, recent results suggest that LRP-1 may facilitate the development and growth of cancer metastases in vivo, but the precise contribution of the receptor during cancer progression remains to be elucidated. The lack of mechanistic insights into the intracellular signaling networks downstream of LRP-1 has prevented the understanding of its contribution towards cancer.

Methodology/Principal Findings

Through a short-hairpin RNA-mediated silencing approach, we identified LRP-1 as a main regulator of ERK and JNK signaling in a tumor cell context. Co-immunoprecipitation experiments revealed that LRP-1 constitutes an intracellular docking site for MAPK containing complexes. By using pharmacological agents, constitutively active and dominant-negative kinases, we demonstrated that LRP-1 maintains malignant cells in an adhesive state that is favorable for invasion by activating ERK and inhibiting JNK. We further demonstrated that the LRP-1-dependent regulation of MAPK signaling organizes the cytoskeletal architecture and mediates adhesive complex turnover in cancer cells. Moreover, we found that LRP-1 is tethered to the actin network and to focal adhesion sites and controls ERK and JNK targeting to talin-rich structures.

Conclusions

We identified ERK and JNK as the main molecular relays by which LRP-1 regulates focal adhesion disassembly of malignant cells to support invasion.     

Bmp7 functions via a polarity mechanism to promote cloacal septation

Background

During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood.

Methodology/Principal Findings

We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse.

Conclusion/Significance

Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

JNK activation is responsible for mucus overproduction in smoke inhalation injury

Background

Increased mucus secretion is one of the important characteristics of the response to smoke inhalation injuries. We hypothesized that gel-forming mucins may contribute to the increased mucus production in a smoke inhalation injury. We investigated the role of c-Jun N-terminal kinase (JNK) in modulating smoke-induced mucus secretion.

Methods

We intubated mice and exposed them to smoke from burning cotton for 15 min. Their lungs were then isolated 4 and 24 h after inhalation injury. Three groups of mice were subjected to the smoke inhalation injury: (1) wild-type (WT) mice, (2) mice lacking JNK1 (JNK1-/- mice), and (3) WT mice administered a JNK inhibitor. The JNK inhibitor (SP-600125) was injected into the mice 1 h after injury.

Results

Smoke exposure caused an increase in the production of mucus in the airway epithelium of the mice along with an increase in MUC5AC gene and protein expression, while the expression of MUC5B was not increased compared with control. We found increased MUC5AC protein expression in the airway epithelium of the WT mice groups both 4 and 24 h after smoke inhalation injury. However, overproduction of mucus and increased MUC5AC protein expression induced by smoke inhalation was suppressed in the JNK inhibitor-treated mice and the JNK1 knockout mice. Smoke exposure did not alter the expression of MUC1 and MUC4 proteins in all 3 groups compared with control.

Conclusion

An increase in epithelial MUC5AC protein expression is associated with the overproduction of mucus in smoke inhalation injury, and that its expression is related on JNK1 signaling.     

A Comparison of the Inflammatory and Proteolytic Effects of Dung Biomass and Cigarette Smoke Exposure in the Lung

Rationale

Biomass is the energy source for cooking and heating for billions of people worldwide. Despite their prevalent use and their potential impact on global health, the effects of these fuels on lung biology and function remain poorly understood.

Methods

We exposed human small airway epithelial cells and C57BL/6 mice to dung biomass smoke or cigarette smoke to compare how these exposures impacted lung signaling and inflammatory and proteolytic responses that have been linked with disease pathogenesis.

Results

The in vitro exposure and siRNA studies demonstrated that biomass and cigarette smoke activated ERK to up regulate IL-8 and MMP-1 expression in human airway epithelial cells. In contrast to cigarette smoke, biomass also activated p38 and JNK within these lung cells and lowered the expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Similarly, in the lungs of mice, both biomass and cigarette smoke exposure increased macrophages, activated ERK and p38 and up regulated MMP-9 and MMP-12 expression. The main differences seen in the exposure studies was that mice exposed to biomass exhibited more perivascular inflammation and had higher G-CSF and GM-CSF lavage fluid levels than mice exposed identically to cigarette smoke.

Conclusion

Biomass activates similar pathogenic processes seen in cigarette smoke exposure that are known to result in the disruption of lung structure. These findings provide biological evidence that public health interventions are needed to address the harm associated with the use of this fuel source.     

Prostaglandin E2 reverses aberrant production of an inflammatory chemokine by microglia from Sandhoff disease model mice through the cAMP-PKA pathway

Background

Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK.

Methodology/Principal Findings

In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice.

Conclusions/Significance

We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD.     

Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

Background

Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells.

Methodology/Principal Findings

DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues.

Conclusions/Significance

We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.     

Ethanol Negatively Regulates Hepatic Differentiation of hESC by Inhibition of the MAPK/ERK Signaling Pathway In Vitro

Background

Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood.

Methods

We employed hepatic progenitor cells derived from hESCs to study the impact of ethanol on hepatocyte differentiation by exposure of these progenitor cells to ethanol during hepatocyte differentiation.

Results

We found that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitor cells in a dose-dependent manner. There was also a moderate cell cycle arrest at G1/S checkpoint in the ethanol treated cells, which is associated with a reduced level of cyclin D1 in these cells. Ethanol treatment specifically inhibited the activation of the ERK but not JNK nor the p38 MAP signaling pathway. At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol. Upon evaluating the effects of the inhibitors of these two signaling pathways, we determined that the Erk inhibitor replicated the effects of ethanol on the hepatocyte differentiation and attenuated the WNT/β-catenin signaling, however, inhibitors of WNT only partially replicated the effects of ethanol on the hepatocyte differentiation.

Conclusion

Our results demonstrated that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitors through inhibiting the MAPK/ERK signaling pathway, and subsequently attenuating the WNT signaling pathway. Thus, our finding provides a novel insight into the mechanism by which alcohol regulates cell fate selection of hESC-derived hepatic progenitor cells, and the identified pathways may provide therapeutic targets aimed at promoting liver repair and regeneration during alcoholic injury.