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1.  Esophageal epithelial and mesenchymal cross-talk leads to features of epithelial to mesenchymal transition in vitro 
Experimental cell research  2012;319(6):850-859.
Background
Esophageal fibrosis is a complication of eosinophilic esophagitis (EoE) which has been attributed to both subepithelial fibrosis and to epithelial to mesenchymal transition (EMT), a process by which epithelial cells acquire mesenchymal features. Common to both causes of EoE-fibrosis is the notion that granulocyte-derived TGF-β, induces myofibroblast differentiation of the target cell. To date, the role of esophageal epithelial cells as effector cells in esophageal fibrosis has never been explored. Here in, we investigated consequences of cross-talk between esophageal epithelial cells and fibroblasts, and identified profibrotic cytokines which influence the development of EMT in vitro.
Methods and Results
Stimulation of primary fetal esophageal fibroblasts (FEF3) with conditioned media (CEM) from esophageal epithelial cells (EPC2-hTERT), primed FEF3 cells to secrete IL-1β and TNFα, but not TGFβ. To determine whether these cytokines signaled in a paracrine fashion to esophageal epithelial cells, FEF3 cells were stimulated with CEM, followed by transfer of this fibroblast conditioned media (FCM) to EPC2-hTERT cells. Epithelial FCM stimulation increased expression of mesenchymal markers and reduced E-cadherin expression, features of EMT which were TNFα and IL-1β-dependent. Using organotypic culture models, primary EoE epithelial cells exhibited features of EMT compared to non-EoE cells, corresponding to patterns of EMT in native biopsies.
Conclusions
Esophageal epithelial cell and fibroblast cross-talk contributes to esophageal fibrosis. Our results suggest that features of EMT can develop in dependent of TGF-β and granulocytes, which may have important implications in treatment of EoE.
doi:10.1016/j.yexcr.2012.12.002
PMCID: PMC3593809  PMID: 23237990
Cross-talk; eosinophilic esophagitis; esophageal epithelial cells; fibroblasts; fibrosis; epithelial to mesenchymal transition; cytokines
2.  Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment 
Experimental cell research  2012;319(6):875-887.
Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancer and severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K-AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Besides caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Over-activation of JNK1 and ERK1/2 and down-regulation of PI3K-AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K-AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through over-activation of MEK-ERK and suppression of PI3K-AKT.
doi:10.1016/j.yexcr.2012.11.019
PMCID: PMC3593966  PMID: 23219847
Neural stem cells; AKT; ERK; JNK; Sodium arsenite
3.  Wnt Interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells 
Experimental cell research  2013;319(6):810-819.
Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1–positive structures appeared in three sizes (small, ≤ 40 nm; intermediates ~40–80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1–containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.
doi:10.1016/j.yexcr.2013.01.003
PMCID: PMC3594006  PMID: 23318676
prominin-1; melanoma; Wnt; catenin; vesicles
4.  Phosphorylation of connexin43 on S279/282 may contribute to laminopathy-associated conduction defects 
Experimental cell research  2012;319(6):888-896.
An understanding of the molecular mechanism behind the arrhythmic phenotype associated with laminopathies has yet to emerge. A-type lamins have been shown to interact and sequester activated phospho-ERK1/2(pERK1/2) at the nucleus. The gap junction protein connexin43 (Cx43) can be phosphorylated by pERK1/2 on S279/282 (pS279/282), inhibiting intercellular communication. We hypothesized that without A-type lamins, pS279/282Cx43 will increase due to inappropriate phosphorylation by pERK1/2, resulting in decreased gap junction function. We observed a 1.6-fold increase in pS279/282 Cx43 levels in Lmna−/− mouse embryonic fibroblasts (MEFs) compared to Lmna+/+, and 1.8-fold more pERK1/2 co-precipitated from Lmna−/− MEFs with Cx43 antibodies. We found a 3-fold increase in the fraction of non-nuclear pERK1/2 and a concomitant 2-fold increase in the fraction of pS279/282 Cx43 in Lmna−/− MEFs by immunofluorescence. In an assay of gap junctional function, Lmna−/− MEFs transferred dye to 60% fewer partners compared to Lmna+/+ controls. These results are mirrored in 5–6 week-old Lmna−/− mice compared to their Lmna+/+ littermates as we detect increased pS279/282 Cx43 in gap junctions by immunofluorescence and 1.7-fold increased levels by immunoblot. We conclude that increased pS279/282 Cx43 in the Lmna−/− background results in decreased cell communication and may contribute to the arrhythmic pathology in vivo.
doi:10.1016/j.yexcr.2012.12.014
PMCID: PMC3594536  PMID: 23261543
nuclear lamins; pERK1/2; connexin43; gap junction; cardiac conduction
5.  Requirement for protein kinase A in the phosphorylation of the TGFß receptor-interacting protein km23-1 as a component of TGFß downstream effects 
Experimental cell research  2013;319(6):897-907.
km23-1 was previously identified as a TGFß-receptor interacting protein that was phosphorylated on serines after TGFß stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFß/ protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFß stimulation. Taken together, our results demonstrate that TGFß induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFß stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFß regulates complex formation between the PKA regulatory subunit RIß and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFß induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFß, with S73-km23-1 being required for the CRE-dependent TGFß stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFß/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.
doi:10.1016/j.yexcr.2012.12.029
PMCID: PMC3723345  PMID: 23333499
TGFß; km23-1; protein kinase A; phosphorylation; signal transduction
6.  Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-beta responsiveness 
Experimental cell research  2007;314(5):1094-1104.
Background
The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance pro-fibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2−/−) mouse embryos.
Methods
The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2−/− MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays.
Results
Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2−/− MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2−/− MEFs, whereas stimulation of alpha-smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2.
Conclusion
Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.
doi:10.1016/j.yexcr.2007.12.010
PMCID: PMC3963386  PMID: 18201696
TGF-β; CTGF/CCN2; fibrosis; fibroblast; Type I collagen
7.  CD147 and AGR2 expression promote cellular proliferation and metastasis of Head and Neck Squamous Cell Carcinoma 
Experimental cell research  2012;318(14):1788-1798.
The signaling pathways facilitating metastasis of head and neck squamous cell carcinoma (HNSCC) cells are not fully understood. CD147 is a transmembrane glycoprotein known to induce cell migration and invasion. AGR2 is a secreted peptide also known to promote cell metastasis. Here we describe their importance in the migration and invasion of HNSCC cells (FADU and OSC-19) in vitro and in vivo. In vitro, knockdown of CD147 or AGR2 decreased cellular proliferation, migration and invasion. In vivo, knockdown of CD147 or AGR2 expression decreased primary tumor growth as well as regional and distant metastasis.
doi:10.1016/j.yexcr.2012.04.022
PMCID: PMC3951318  PMID: 22659167
Aerodigestive squamous cell carcinoma; head and neck; metastasis; cd147
8.  Epigallocatechin Gallate and Sulforaphane Combination Treatment Induce Apoptosis in Paclitaxel-Resistant Ovarian Cancer Cells through hTERT and Bcl-2 Down-regulation 
Experimental cell research  2013;319(5):697-706.
The cellular development of resistance to chemotherapy contributes to the high mortality noted in patients affected by ovarian cancer. Novel compounds that specifically target cellular drug resistance in ovarian cancer are therefore highly desired. Previous epidemiological studies indicate that consumption of green tea and cruciferous vegetables is inversely associated with occurrence of ovarian cancer. Therefore revealing the effects and mechanisms of major components of green tea (epigallocatechin gallate, EGCG) and cruciferous vegetables (sulforaphane, SFN) on ovarian cancer cells will provide necessary knowledge for developing potential novel treatments for the disease. In this study, EGCG or SFN was used to treat both paclitaxel-sensitive (SKOV3-ip1) and -resistant (SKOV3TR-ip2) ovarian cancer cell lines alone or in combination. We found that SFN inhibits cell viability of both ovarian cancer cell lines time- and dose-dependently and that EGCG potentiates the inhibiting effect of SFN on ovarian cancer cells. Cell cycle analysis indicates SFN can arrest ovarian cancer cells in G2/M phase, while EGCG and SFN co-treatment can arrest cells in both G2/M and S phase. Combined EGCG and SFN treatment increases apoptosis significantly in paclitaxel-resistant SKOV3TR-ip2 cells after 6 days of treatment, while reducing the expression of hTERT, the main regulatory subunit of telomerase. Western blotting also indicates that SFN can down-regulate Bcl-2 (a gene involved in anti-apoptosis) protein levels in both cell types. Cleaved poly(ADP-ribose) polymerase (PARP) becomes up-regulated by 6 days of treatment with SFN and this is more pronounced for combination treatment indicating induction of apoptosis. Furthermore, phosphorylated H2AX is up-regulated after 6 days of treatment with SFN alone, and EGCG can potentiate this effect, suggesting that DNA damage is a potential cellular mechanism contributing to the inhibiting effect of EGCG and SFN combination treatment. Taken together, these results indicate that EGCG and SFN combination treatment can induce apoptosis by down-regulating of hTERT and Bcl-2 and promote DNA damage response specifically in paclitaxel-resistant ovarian cancer cell lines and suggest the use of these compounds for overcoming paclitaxel resistance in ovarian cancer treatment.
doi:10.1016/j.yexcr.2012.12.026
PMCID: PMC3577973  PMID: 23333498
ovarian cancer; SKOV3; epigallocatechin gallate; sulforaphane; paclitaxel
9.  Cyclophilin 40 alters UVA-induced apoptosis and mitochondrial ROS generation in keratinocytes 
Experimental cell research  2012;319(5):750-760.
The CyP40 protein encoded by PPID gene is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. The CyP40 protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant drug cyclosporin A (CsA). In this study, we created keratinocyte cell lines with CyP40 being stably knocked down using viral particles containing shRNA for CyP40 which knocked down the expression level of CyP40 transcripts by 90 to 99%. The proliferation rates of the cell lines with silenced CyP40 were decreased compared to the control cells. After UVA irradiation, the rate of apoptosis was found to be significantly lower in CyP40 silenced cell lines than it was in control cells. Moreover, mitochondrial membrane potential (MMP) was found to be less dissipated and mitochondrial permeability transition pore (MPTP) less active in cells with knocked down CyP40 than in control cells after UVA irradiation. Also, less mitochondrial superoxide was detected in the cells with silenced CyP40 compared to control cells after UVA exposure. Moreover, silencing of CyP40 partially modulates expression of key genes involved in mitochondrial pore formation including CyPD, ANTs and VDAC family members. The ability of CyP40 to regulate UV induced apoptosis implicates this protein as a potential target for therapy in cancer cells.
doi:10.1016/j.yexcr.2012.11.016
PMCID: PMC3577976  PMID: 23220213
CyP40; stable CyP40 knock-down; mitochondrial membrane potential; mitochondrial pore opening; UVA-induced apoptosis; reactive oxygen species; keratinocytes
10.  Ubiquitin specific protease 18 (Usp18) is a WT1 transcriptional target 
Experimental cell research  2013;319(5):612-622.
Wilms tumor gene WT1 encodes a zinc finger containing transcription factor which is required for renal development. Mutations in WT1 are observed in 20% of Wilms tumor (a pediatric kidney cancer), but the in vivo WT1 targets and associated molecular pathways involved in the etiology of Wilms tumor are still elusive. To identify WT1 targets we performed genome wide comprehensive expression profiling using Affymetrix Gene Chip Mouse Genome 430 2.0 Array, comparing E13.5 mouse kidneys in which Wt1 had been somatically ablated with littermate controls. We identified Usp18 as the most differentially expressed gene in mutant kidney. Using tetracycline inducible cells we demonstrated a repressive effect of WT1 on USP18 expression. Conversely, knockdown of WT1 led to the upregulation of Usp18. Furthermore, direct binding of WT1 to the Usp18 promoter was demonstrated by ChIP assay. Overexpression of USP18 in murine and human cell lines resulted in cell proliferation. Additionally, Usp18 upregulation was observed in a mouse model of Wilms tumor. Taken together our data demonstrate that Usp18 is a transcriptional target of WT1 and suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis.
doi:10.1016/j.yexcr.2012.12.021
PMCID: PMC3791519  PMID: 23291318
USP18; WT1; Wilms tumor; Transcription; Target
11.  Specific contribution of lamin A and lamin C in the development of laminopathies 
Experimental cell research  2008;314(13):2362-2375.
Mutations in the lamin A/C gene are involved in multiple human disorders for which the pathophysiological mechanisms are partially understood. Conflicting results prevail regarding the organization of lamin A and C mutants within the nuclear envelope (NE) and on the interactions of each lamin to its counterpart. We over-expressed various lamin A and C mutants both independently and together in COS7 cells. When expressed alone, lamin A with cardiac/muscular disorder mutations forms abnormal aggregates inside the NE and not inside the nucleoplasm. Conversely, the equivalent lamin C organizes as intranucleoplasmic aggregates that never connect to the NE as opposed to wild type lamin C. Interestingly, the lamin C molecules present within these aggregates exhibit an abnormal increased mobility. When co-expressed, the complex formed by lamin A/C aggregates in the NE. Lamin A and C mutants for lipodystrophy behave similarly to the wild type. These findings reveal that lamins A and C may be differentially affected depending on the mutation. This results in multiple possible physiological consequences which likely contribute in the phenotypic variability of laminopathies. The inability of lamin C mutants to join the nuclear rim in the absence of lamin A is a potential pathophysiological mechanism for laminopathies.
doi:10.1016/j.yexcr.2008.04.017
PMCID: PMC3934841  PMID: 18538321 CAMSID: cams3911
Lamin A/C gene; Laminopathy; Nuclear envelope; FRAP; Electron microscopy
12.  Autophagy is Required for IL-2-Mediated Fibroblast Growth 
Experimental cell research  2012;319(4):556-565.
Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.
doi:10.1016/j.yexcr.2012.11.012
PMCID: PMC3563717  PMID: 23195496
IL-2; Autophagy; Apoptosis; Immunotherapy; HMGB1
13.  Contributions of microRNA Dysregulation to Cisplatin Resistance in Adenocarcinoma Cells 
Experimental cell research  2012;319(4):566-574.
Cisplatin resistance in cancer cells is due to a pleiotropic phenotype transition that allows cells to resist cell death. miRNAs have been shown to be reliable markers of phenotype, critical in cell differentiation, and dysregulated in cancer and other pathologies. Here we investigate the influence of miRNA on cisplatin resistance in KB adenocarcinoma cells. Silencing both DICER and TRBP2 in the miRNA biosynthesis pathway in KB-3-1 (sensitive parental), KB-CP.5 (cisplatin-resistant), and KB-CP20 (highly cisplatin-resistant) cells resulted in the reversal of cisplatin resistance, with no effect on cell viability in the absence of cisplatin. We found miR-181 expression differences in the cell lines using RT-PCR, with several members of the miR-181 family overexpressed in two KB cisplatin-resistant lines and in two cisplatin-resistant lung cancer lines, compared to their respective parental cells. Functional assays showed minimal effects of miR-181 on cisplatin resistance. We conclude that the miRNA biosynthesis pathway is critical for maintaining the cisplatin-resistant phenotype, but that it is difficult to determine the precise miRNAs involved in cisplatin resistance simply using expression profiles of individual miRNA species. Functional assays are needed to determine the influence of a specific miRNA and different members of the same miRNA family may have opposite effects.
doi:10.1016/j.yexcr.2012.10.012
PMCID: PMC3563763  PMID: 23137650
Drug resistance; cisplatin; microRNA; chemotherapy; drug selection
14.  Fibroblast Cluster Formation on 3D Collagen Matrices Requires Cell Contraction-Dependent Fibronectin Matrix Organization 
Experimental cell research  2012;319(4):546-555.
Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy.
doi:10.1016/j.yexcr.2012.10.005
PMCID: PMC3563764  PMID: 23117111
3D collagen matrix; Fibronectin; Integrin; Cell migration; Cell contraction; Wound repair; Tissue morphogenesis
15.  Regulation of MEF2 Transcriptional Activity by Calcineurin/mAKAP Complexes 
Experimental cell research  2012;319(4):447-454.
The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity. We now show that the scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2 association is dependent on mAKAP expression. We have identified a peptide comprising the calcineurin binding domain in mAKAP that can disrupt the binding of the phosphatase to the scaffold in vivo. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together these data illustrate the importance of calcineurin anchoring by the mAKAP scaffold for MEF2 regulation.
doi:10.1016/j.yexcr.2012.12.016
PMCID: PMC3563847  PMID: 23261540
Scaffold; AKAP; protein phosphatase; Calcineurin; MEF2
16.  WNK kinases and the kidney 
Experimental cell research  2012;318(9):1020-1026.
In the kidney, the renal tubule plays a major role in maintaining fluid and electrolyte balance. This balance is achieved by an interplay between various hormones and nerves that signal changes throughout the body and transfer these signals to transport proteins. Increased or reduced activity of these transporters helps to restore homeostasis, but can also contribute to disease (e.g. sodium retention in hypertension). In recent years, it has become clear that the signal transfer to transporters is largely mediated by kinases. Among these, WNK kinases (With No lysine = K) stand out, because they regulate the major sodium and potassium transporters in the distal nephron. Moreover, mutations in genes encoding WNK kinases result in an inherited form of salt-sensitive hypertension with hyperkalemia, illustrating their important role in sodium, potassium, and blood pressure regulation. More recently, WNK kinases were found to play a role in acquired forms of hypertension as well. Together, the evolving insight in the kinase regulation of ion transport is providing new insights in the longstanding question how salt and blood pressure are related. Here, we review the current models of how WNK kinases regulate the various transport proteins and which roles they play in health and disease.
doi:10.1016/j.yexcr.2012.02.029
PMCID: PMC3922210  PMID: 22405999
Distal tubule; WNK kinases; Hypertension; NaCl transport; SPAK; Pseudohypoaldosteronism type 2
17.  The Activation of Beta-Catenin by Wnt Signaling Mediates the Effects of Histone Deacetylase Inhibitors 
Experimental cell research  2007;313(8):1652-1666.
Most colorectal carcinomas (CRCs) exhibit constitutively active Wnt signaling. We have reported that (a) the histone deacetylase inhibitor (HDACi)2 sodium butyrate (NaB) modulates the canonical Wnt transcriptional activity of CRC cells in vitro and (b) a linear relationship exists between the increase in Wnt transcriptional activity and the levels of apoptosis in ten CRC cell lines treated with NaB. Herein we report that structurally different HDACis modulate Wnt signaling in CRC cells and a mechanism involved in this action is an increase in beta-catenin that is dephosphorylated at Ser-37 and Thr-41 residues. The increase of active (Ser-37 and Thr-41 dephosphorylated) beta-catenin in CRC cells treated with HDACis is initiated at the ligand level and the inhibition of this increase suppresses Wnt signaling and lowers the levels of apoptosis. CRC cells that develop resistance to the apoptotic effects of HDACis exhibit lower levels of active beta-catenin compared to apoptosis-sensitive parental cells and this resistance is reversed by increasing the levels of active beta-catenin. Results from comparative studies between HDACi-resistant and HDACi-sensitive cells suggest that non-histone targets of HDACis mediate the effects on Wnt signaling and apoptosis.
doi:10.1016/j.yexcr.2007.02.008
PMCID: PMC3919021  PMID: 17359971
Wnt signaling; histone deacetylase inhibitors; apoptosis; colorectal carcinomas; butyrate
18.  Development transitions of thin filament proteins in rat extraocular muscles 
Experimental cell research  2012;319(3):23-31.
Extraocular muscles are a unique subset of striated muscles. During postnatal development, the extraocular muscles undergo a number of myosin isoform transitions that occur between postnatal day 10 (P10) and P15. These include: 1) loss of embryonic myosin from the global layer resulting in the expression restricted to the orbital layer; 2) the onset of expression of extraocular myosin and the putative tonic myosin (myh 7b/14); and 3) the redistribution of nonmuscle myosin IIB from a subsarcolemma position to a sarcomeric distribution in the slow fibers of the global layer. For this study, we examined the postnatal appearance and distribution of α-actinin, tropomyosin, and nebulin isoforms during postnatal development of the rat extraocular muscles. Although sarcomeric α-actinin is detectable from birth, α-actinin 3 appears around P15. Both tropomyosin-1 and -2 are present from birth in the same distribution as in the adult animal. The expression of nebulin was monitored by gel electrophoresis and western blots. At P5–10, nebulin exhibits a lower molecular mass than observed P15 and later during postnatal development. The changes in α-actinin3 and nebulin expression between P10 and 15 coincide with transitions in myosin isoforms as detailed above. These data point to P10–P15 as the critical period for the maturation of the extraocular muscles, coinciding with eyelid opening.
doi:10.1016/j.yexcr.2012.11.010
PMCID: PMC3546233  PMID: 23174654
19.  IFT80 is essential for chondrocyte differentiation by regulating hedgehog and Wnt signaling pathways 
Experimental cell research  2013;319(5):623-632.
Partial mutation of intraflagellar transport 80 (IFT80) in humans causes Jeune asphyxiating thoracic dystrophy (JATD) and short-rib polydactyly (SRP) syndrome type III. These diseases are autosomal recessive chondrodysplasias that share clinical similarities, including shortened long bones and constricted thoracic cage. However, the role and mechanism of IFT80 in the regulation of chondrocyte differentiation and function remain largely unknown. We hypothesize that IFT80 is required for the formation and function of cilia and plays a critical role in chondrogenic differentiation by regulating Hedgehog (Hh) and Wingless (Wnt) signaling pathways. To test this hypothesis, we first analyzed the IFT80 expression pattern and found that IFT80 was predominantly expressed in growth plate chondrocytes and during chondrogenic differentiation. Silencing IFT80 impaired cilia formation and chondrogenic differentiation in mouse bone marrow derived stromal cells (BMSCs), and decreased the expression of chondrocyte marker genes—collagen II and aggrecan. Additionally, silencing IFT80 down-regulated Hh signaling activity whereas up-regulated Wnt signaling activity. The overexpression of Gli2 in IFT80-silenced cells promoted chondrogenesis and recovered the chondrogenic deficiency from IFT80 silencing. Overall, our results demonstrate that IFT80 is essential for chondrocyte differentiation by regulating the Hh and Wnt signaling pathways.
doi:10.1016/j.yexcr.2012.12.028
PMCID: PMC3908790  PMID: 23333501
Intraflagellar transport; Cilia; Chondrogenic differentiation; Hedgehog signaling; Wnt signaling
20.  Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates 
Experimental cell research  2005;311(2):10.1016/j.yexcr.2005.10.007.
During embryonic neural development, axon tips (“growth cones”) are guided through a dynamic three-dimensional (3-D) landscape by soluble chemotropic factors and by immobilized, growth-permissive or growth-inhibiting contact cues present in the extracellular matrix and on the surface of surrounding cells. It has been difficult to probe the search algorithms of growth cones in response to multiple contact cues during 3-D navigation using traditional two-dimensional (2-D) substrates. Here, we present an in vitro study in which the axons of murine embryonic cortical neurons are challenged with competing growth options, using 3-D substrates that feature variations in permissiveness and microtopography. As 3-D substrates, we used poly-d-lysine (PDL) coatings on microfabricated steps of polydimethylsiloxane (PDMS) and complementary features of Matrigel. We found that axons display a preference for PDL over Matrigel and for the straightest path within a distance consistent with the exploratory range of the growth cone. When these two preferences are in conflict, axons choose to grow straight into Matrigel; when the straight path is not permissive, the axon turns in the direction that minimizes the turning angle. These results suggest that growth cones make 3-D navigation decisions by integrating permissiveness and topographical cues.
doi:10.1016/j.yexcr.2005.10.007
PMCID: PMC3880801  PMID: 16263111
Axon guidance; Cell culture; Embryonic; PDMS; Matrigel; Surface micropatterning; Surface microstructure
21.  Skeletal muscle neural progenitor cells exhibit properties of NG2-glia 
Experimental cell research  2012;319(1):45-63.
Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice, but their identity, origin, and potential survival in the brain are only vaguely understood. In this work, we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia, including NG2, PDGFRα, O4, NGF receptor (p75), glutamate receptor-1(AMPA), and A2B5 expression. Although these cells exhibit NG2, they do not express other pericyte markers, such as α-SMA or connexin-43, and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents, probably carried through Kir6.1 channels. Given their potential therapeutic application, we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes, we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan.
doi:10.1016/j.yexcr.2012.09.008
PMCID: PMC3597239  PMID: 22999866
Nestin-expressing neural progenitors; NG2-glia; Stem cells; Skeletal muscle; Progenitor cells; Nestin
22.  Valosin containing protein (VCP/p97) is a novel substrate for the protein tyrosine phosphatase PTPL1 
Experimental cell research  2012;319(1):1-11.
Identification of Protein Tyrosine Phosphatase (PTP) substrates is critical in understanding cellular role in normal cells as well as cancer cells. We have previously shown that reduction of PTPL1 protein levels in Ewings sarcoma (ES) inhibit cell growth and tumorigenesis. Therefore, we sought to identify novel PTPL1 substrates that may be important for tumorigenesis. In this current work, we demonstrated that mouse embryonic fibroblasts without PTPL1 catalytic activity fail to form foci when transfected with oncogenes. We proved that catalytic activity of PTPL1 is important for ES cell growth. Using a substrate-trapping mutant of PTPL1 we identified putative PTPL1 substrates by mass-spectrometry. One of these putative substrates was characterized as Valosin Containing Protein (VCP/p97). Using multiple biochemical assays we validated VCP as a novel substrate of PTPL1. We also provide evidence that tyrosine phosphorylation of VCP might be important for its midbody localization during cytokinesis. In conclusion, our work identifies VCP as a new substrate for PTPL1, which may be important in cellular transformation. Our investigation link an oncogenic transcription factor EWS-FLI1, with a key transcriptional target protein tyrosine phosphatase PTPL1, and its substrate VCP. Given our observation that PTPL1 catalytic activity is important for cell transformation, our results may also suggest that VCP regulation by PTPL1 might be important for tumorigenesis.
doi:10.1016/j.yexcr.2012.09.003
PMCID: PMC3638858  PMID: 23018179
ES; PTPL1; VCP; Phosphorylation
23.  Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry 
Experimental cell research  2009;315(10):10.1016/j.yexcr.2009.03.006.
Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry.
Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.
doi:10.1016/j.yexcr.2009.03.006
PMCID: PMC3874874  PMID: 19298813
SYTO; Apoptosis; Kinetic assays; Antitumor drugs; Microfluidics; Lab-on-a-Chip; Flow cytometry
24.  CTCF Mediates Effect of Insulin On Glucagon Expression 
Experimental cell research  2012;318(8):10.1016/j.yexcr.2012.03.001.
Pancreatic islet α-cell development and glucagon production are mainly regulated by Pax6 in the homeobox gene families. However, the molecular mechanism fine-tuning the regulation of these events in α-cell still remains unclear. In ocular cells, Pax6 transcription is regulated by CTCF through its binding to specific sites in Pax6 promoter. In this study, CTCF-mediated regulations of islet α-cell development and glucagon production were investigated in both CTCF transgenic mice and α-TC-1-6 cells. Over-expression of CTCF in transgenic mice affected development of pancreatic islets by significantly suppressing α-cell population in both embryonic and adult pancreases. The effect of CTCF on Pax6 gene expression and subsequently, on pro-glucagon production was however, examined in pancreatic islet α-cells. Over-expression and knock-down of CTCF directly affected Pax6 expression. More importantly, the CTCF binding sites upstream from Pax6 p0 promoter were required for regulating p0 promoter activity in islet α-cells. Stimulation of α-cells with insulin resulted in a significant increase in CTCF expression and a decrease in Pax6 expression, and consequently suppressed pro-glucagon expression. In contrast, these insulin-induced effects were blocked by knockdown of CTCF mRNA with specific siRNA in α-cells. Altogether, our results demonstrated for the first time that CTCF functions as a switch-like molecule between the insulin signaling and the regulations of Pax6 and glucagon expression in pancreatic islet α-cells.
doi:10.1016/j.yexcr.2012.03.001
PMCID: PMC3875169  PMID: 22426149
Pax6; gene regulation; insulin; islet cells; gene transcription; α-cell
25.  Sterol-Dependent Nuclear Import of ORP1S Promotes LXR Regulated Trans-Activation of APOE 
Experimental cell research  2012;318(16):10.1016/j.yexcr.2012.06.012.
Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to the nucleus and promotes LXR-dependent gene transcription through select enhancer elements.
doi:10.1016/j.yexcr.2012.06.012
PMCID: PMC3867128  PMID: 22728266
ORP1; ORP1S; oxysterol; LXR; nuclear import; NLS

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