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.
ORP1; ORP1S; oxysterol; LXR; nuclear import; NLS
Neurokinin 1 (NK1) encodes full-length (NK1-FL) and truncated (NK1-Tr) receptors, with distinct 3′ UTR. NK1-Tr exerts oncogenic functions and is increased in breast cancer (BC). Enhanced transcription of NK1 resulted in higher level of NK1-Tr. The 3′ UTR of these two transcripts are distinct with NK1-Tr terminating at a premature stop codon. NK1-Tr mRNA gained an advantage over NK1-FL with regards to translation. This is due to the ability of miR519B to interact with sequences within the 3′ UTR of NK1-FL, but not NK1-Tr since the corresponding region is omitted. MiR519b suppressed the translation of NK1-FL in T47D and MDA-MB-231 resulting increased NK1-Tr protein. Cytokines can induce the transcription of NK1. However, our studies indicated that translation appeared to be independent of cytokine production by the BC cells (BCCs). This suggested that transcription and translation of NK1 might be independent. The findings were validated in vivo. MiR-519b suppressed the growth of MDA-MB-231 in 7/10 nude BALB/c. In total, increased NK1-Tr in BCCs is due to enhanced transcription and suppressed translation of NK1-FL by miR-519b to reduced tumor growth. In summary, we report on miRNA as a method to further regulate the expression of a spiced variant to promote oncogenesis. In addition, the findings have implications for therapy with NK1 antagonists. The oncogenic effect of NK1-Tr must be considered to improve the efficacy of current drugs to NK1.
Neurokinin-1; miR519b; breast cancer; translation; transcription
Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) of potential use in patients with breast cancer. Unfortunately, in clinical studies, gefitinib is often ineffective indicating that resistance to EGFR inhibitors may be a common occurrence in cancer of the breast. EGFR has been shown to be overexpressed in breast cancer, and in particular remains hyperphosphorylated in cell lines such as MDA-MB-468 that are resistant to EGFR inhibitors. Here, we investigate the cause of this sustained phosphorylation and the molecular basis for the ineffectiveness of gefitinib. We show that reactive oxygen species (ROS), known to damage cellular macromolecules and to modulate signaling cascades in a variety of human diseases including cancers, appear to play a critical role in mediating EGFR TKI-resistance. Furthermore, elimination of these ROS through use of a cell-penetrating catalase derivative sensitizes the cells to gefitinib. These results suggest a new approach for the treatment of TKI-resistant breast cancer patients specifically, the targeting of ROS and attendant downstream oxidative stress and their effects on signaling cascades.
Breast cancer; Reactive oxygen species; Tyrosine kinase inhibitors; Epidermal growth factor receptor; Tyrosine kinase
Sarcospan is a component of the dystrophin-glycoprotein complex that forms a tight subcomplex with the sarcoglycans. The sarcoglycan-sarcospan subcomplex functions to stabilize α-dystroglycan at the plasma membrane and perturbations of this subcomplex are associated with autosomal recessive limb-girdle muscular dystrophy. In order to characterize protein interactions within this subcomplex, we first demonstrate that sarcospan forms homo-oligomers within the membrane. Experiments with a panel of site-directed mutants reveal that proper structure of the large extracellular loop is an important determinant of oligo formation. Furthermore, the intracellular N- and C-termini contribute to stability of sarcospan-mediated webs. Point mutation of each cysteine residue reveals that Cys 162 and Cys 164 within the large extracellular loop form disulfide bridges, which are critical for proper sarcospan structure. The extracellular domain of sarcospan also forms the main binding site for the sarcoglycans. We propose a model whereby sarcospan forms homo-oligomers that cluster the components of the dystrophin-glycoprotein complex within the membrane.
sarcospan; sarcoglycan; dystrophin-glycoprotein complex; tetraspanin; oligomerization
All-trans-retinoic acid (RA), a natural metabolite of retinol, carries out most of the biological activities of vitamin A and is required for normal growth, cell differentiation, and immune functions. In the present studies, THP-1 human monocytes were used to investigate the mechanisms by which RA may regulate progression through the G1/S phase of the cell cycle. Physiological concentrations of all-trans-RA reduced the levels of cyclin E mRNA by 6 h and reduced cyclin E protein in a dose- and time-dependent manner. Similar reductions were observed for the retinoic acid receptor RARα and RXRα proteins. Concomitantly, RA increased the level of the cyclin-dependent kinase inhibitor p27 (Kip-1). The levels of retinoblastoma mRNA and protein (pRb) were also increased, while the proportion of hyperphosphorylated (phosphoserine 807/811) pRb was markedly reduced. Overall, RA increased the functionality of pRb as an inhibitor of cell cycle progression. Furthermore, RA reduced the binding activity of the transcription factor E2F to its core DNA element. Retinoic acid-induced changes in cell cycle-related proteins occurred in 4–6 h, including reduced cyclin E expression in bromodeoxyuridine (BrdU)-labeled cells, before the onset of cell differentiation as indicated by an increase in the percentage of G1 phase cells and a reduction in S phase cells at 24 h. The expression of CD11b, a cell surface marker of macrophage-like differentiation was increased by RA, as was phagocytic activity. The multiple effects of RA on cell cycle progression may help to explain its well-documented ability to induce the differentiation of THP-1 cells, and thereby to enhance macrophage-like immune functions.
Cyclin E; Retinoblastoma protein; p27; CD11b; Gene expression; Retinoic acid
In the bone marrow cavity, hematopoietic stem cells (HSC) have been shown to reside in the endosteal and subendosteal perivascular niches, which play specific roles on HSC maintenance. Although cells with long-term ability to reconstitute full hematopoietic system can be isolated from both niches, several data support a heterogenous distribution regarding the cycling behavior of HSC. Whether this distinct behavior depends upon the role played by the stromal populations which distinctly create these two niches is a question that remains open. In the present report, we used our previously described in vivo assay to demonstrate that endosteal and subendosteal stromal populations are very distinct regarding skeletal lineage differentiation potential. This was further supported by a microarray-based analysis, which also demonstrated that these two stromal populations play distinct, albeit complementary, roles in HSC niche. Both stromal populations were preferentially isolated from the trabecular region and behave distinctly in vitro, as previously reported. Even though these two niches are organized in a very close range, in vivo assays and molecular analyses allowed us to identify endosteal stroma (F-OST) cells as fully committed osteoblasts and subendosteal stroma (F-RET) cells as uncommitted mesenchymal cells mainly represented by perivascular reticular cells expressing high levels of chemokine ligand, CXCL12. Interestingly, a number of cytokines and growth factors including interleukin-6 (IL-6), IL-7, IL-15, Hepatocyte growth factor (HGF) and stem cell factor (SCF) matrix metalloproteases (MMPs) were also found to be differentially expressed by F-OST and F-RET cells. Further microarray analyses indicated important mechanisms used by the two stromal compartments in order to create and coordinate the “quiescent” and “proliferative” niches in which hematopoietic stem cells and progenitors reside.
bone marrow; trabecular bone; microenvironment; osteoblast; niche
Mesenchymal stem cells (MSCs) represent a promising cellular therapeutic for the treatment of a variety of disorders. On transplantation, MSCs interact with diverse extracellular matrices (ECMs) that vary dramatically in topographic feature type, size and surface order. In order to investigate the impact of these topographic cues, surfaces were fabricated with either isotropically ordered holes or anisotropically ordered ridges and grooves. To simulate the biologically relevant nano through micron size scale, a series of topographically patterned substrates possessing features of differing pitch (pitch=feature width+groove width) were created. Results document that the surface order and size of substratum topographic features dramatically modulate fundamental MSC behaviors. Topographically patterned (ridge+groove) surfaces were found to significantly impact MSC alignment, elongation, and aspect ratio. Novel findings also demonstrate that submicron surfaces patterned with holes resulted in increased MSC alignment to adjacent cells as well as increased migration rates. Overall, this study demonstrates that the presentation of substratum topographic cues dramatically influence MSC behaviors in a size and shape dependent manner. The response of MSCs to substratum topographic cues was similar to other cell types that have been studied previously with regards to cell shape on ridge and groove surfaces but differed with respect to proliferation and migration. This is the first study to compare the impact of anisotropically ordered ridge and groove topographic cues to isotropically order holed topographic cues on fundamental MSC behaviors across a range of biologically relevant size scales.
Mesenchymal stem cells; Topography; Canine; Nuclear alignment; Migration
All-trans-retinoic acid (RA), the major active metabolite of vitamin A, is a regulator of gene expression with many roles in cell differentiation. In the present study, we investigated RA in the regulation of MafB, a basic leucine-zipper transcription factor with broad roles in embryonic development, hematopoiesis and monocyte-macrophage differentiation. In RA-treated THP-1 human monocytic cells, MafB mRNA and protein levels were up-regulated by RA dose and time-dependently, while, additionally, RA and tumor necrosis factor (TNF)α, also known to induce monocyte to macrophage differentiation, increased MafB expression synergistically. Screening of potential targets containing Maf recognition elements (MARE motifs) in their promoter regions identified SPOCK1, Blimp1 and CCL2 as potential targets; these genes are related to cell communication, recruitment and differentiation, respectively. Across cell treatments, SPOCK1, Blimp1 and CCL2 mRNA levels were highly correlated (P<0.001) with MafB. ChIP assays demonstrated increased MafB protein binding to MARE elements in the promoter regions of SPOCK1, Blimp1 and CCL2 in RA and TNFα-treated cells, as well as acetylation of histone-H4 in MARE-containing regions, indicative of chromatin activation. Conversely, reducing MafB protein by microRNA silencing significantly decreased the expression of SPOCK1, Blimp1 and CCL2 (P<0.01). Moreover, the reduction in MafB expression and these downstream targets correlated with decreased cell differentiation as determined by cell-surface CD11b expression and phagocytic activity. We conclude that MafB may be a key factor in mediating the ability of RA and TNFα to regulate monocytic cell communication, recruitment and differentiation through regulation of MafB target genes including SPOCK1, CCL2 and Blimp1.
retinoic acid; MafB; MafB target genes; MafB silencing; monocytic cell differentiation
We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-κB (NF-κB) is a key transcription factor for production of MMPs. An inhibitor of NF-κB activation, Bay11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-κB in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids.
mtDNA mutation; reactive oxygen species; microarrays; gene expression; MMP-9; migration and invasion
The fundamental question of how and which neuronal specific transcription factors tailor mitochondrial bioenergetics to the need of developing neuronal cells has remained largely unexplored. In this study, we report that the neurogenic basic helix-loop-helix transcription factor NeuroD6 possesses mitochondrial biogenic properties by amplifying the mitochondrial DNA content and TFAM expression levels, a key regulator for mitochondrial biogenesis. NeuroD6-mediated increase in mitochondrial biogenesis in the neuronal progenitor-like PC12-NEUROD6 cells is concomitant with enhanced mitochondrial bioenergetic functions, including increased expression levels of specific subunits of respiratory complexes of the electron transport chain, elevated mitochondrial membrane potential and ATP levels produced by oxidative phosphorylation. Thus, NeuroD6 augments the bioenergetic capacity of PC12-NEUROD6 cells to generate an energetic reserve, which confers tolerance to the mitochondrial stressor, rotenone. We found that NeuroD6 induces an adaptive bioenergetic response throughout rotenone treatment involving maintenance of the mitochondrial membrane potential and ATP levels in conjunction with preservation of the actin network. In conclusion, our results support the concept that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal differentiation with acquisition of adequate mitochondrial mass and energetic capacity to ensure energy demanding events, such as cytoskeletal remodeling, plasmalemmal expansion, and growth cone formation.
NeuroD family; neuronal differentiation; mitochondrial biogenesis; mitochondrial membrane potential; oxidative phosphorylation; mitochondrial stressors
We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.
•RAF and antioxidants show equal protection against ROS and cell death.•Antioxidants prevented BAX activation but not mitochondrial translocation.•No significant impact of antioxidants on Bim or Mcl-1 expression was observed.•ROS did not alter the activity of intracellular signaling pathways.•Bcl-2 proteins are critical for the survival activity of RAF and antioxidants.
RAF; Antioxidants; Reactive oxygen species (ROS); Mitochondria; Bcl-2 proteins; Apoptosis
Endocytic trafficking plays an important role in signal transduction. Signal transducer and activator of transcription 3 (STAT3) has been localized to endosomal structures and is dependent upon endocytosis for downstream function, but the nature of the interaction between STAT3 and endosomes is poorly defined. Interleukin-6 (IL-6) treatment time courses combined with pharmacologic and temperature inhibition, pulse-chase assays, and in vitro kinase assays allow us to evaluate the role of endosomes in the initiation, modulation, amplification and persistence of STAT3 signal transduction and transcription. We demonstrate that IL-6-induced STAT3 activation is initiated by direct interaction with internal structures upstream of the lateendosome and that persistent STAT3 signaling depends upon recurrent activation from endocytic structures. Further, we show that STAT3 subcellular localization is not dependent upon endocytic trafficking. Instead, STAT3 interacts with endosomes transiently and relocates to the nucleus by an endosome-independent mechanism. Finally, we establish STAT3 serine 727 phosphorylation as dependent upon endocytic trafficking and crosstalk with the mitogen-activated protein kinase (MAPK) signaling system. These data reveal endosomes as central to the genesis, course and outcome of STAT3 signal transduction and transcription.
STAT3; MAPK; Erk1/2; IL-6; Signaling Endosome
Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells.
Wound repair; Neural progenitor cells; Dorsal root ganglion neurons; Microvascular endothelial cells; Paracrine interactions; Angiogenesis
Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs.
Embryonic stem cells; cell cycle; self-renewal; pluripotency
Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions.
Brg1; Brm; chromatin remodeling; cisplatin
The capacity to follow cell type-specific signaling in intact lung remains limited. 20-hydroxyeicosatetraenoic acid (20-HETE) is an endogenous fatty acid that mediates signaling for a number of key physiologic endpoints in the pulmonary vasculature, including cell survival and altered vascular tone. We used confocal microscopy to identify enhanced reactive oxygen species (ROS) production in endothelial cell (EC)s in intact lung evoked by two stable analogs of 20-HETE, 20-5,14-HEDE (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid) and 20-5,14-HEDGE (N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine). These analogs generated increased ROS in cultured pulmonary artery endothelial cells as well. 20-HETE analog treatment decreased apoptosis of pulmonary tissue exposed to hypoxia-reoxygenation (HR) ex vivo. Enhanced ROS production and apoptosis were confirmed by biochemical assays. Our studies identify physiologically critical, graded ROS from ECs in live lung tissue ex vivo treated with 20-HETE analogs and protection from HR-induced apoptosis. These methodologies create exciting possibilities for studying signaling by stable 20-HETE analogs and other factors in pulmonary endothelial and other lung cell types in their native milieu.
dihydroethidium; eicosanoid; reactive oxygen species; TUNEL; confocal
Glomerulosclerosis is characterized by excessive deposition of extracellular matrix within the glomeruli of the kidney, glomerular cell death, and subsequent loss of functional glomeruli. While in physiological situations the levels of extracellular matrix components are kept constant by a tight balance between formation and degradation, in the case of injury that results in fibrosis there is increased matrix deposition relative to its breakdown. Multiple factors control matrix synthesis and degradation, thus contributing to the development of glomerulosclerosis. This review focuses primarily on the role of cell-matrix interactions, which play a critical role in governing glomerular cell cues in both healthy and diseased kidneys. Cell-extracellular matrix interactions are made possible by various cellular receptors including integrins, discoidin domain receptors, and dystroglycan. Upon binding to a selective extracellular matrix protein, these receptors activate intracellular signaling pathways that can either downregulate or upregulate matrix synthesis and deposition. This, together with the observation that changes in the expression levels of matrix receptors have been documented in glomerular disease, clearly emphasizes the contribution of cell-matrix interactions in glomerular injury. Understanding the molecular mechanisms whereby extracellular matrix receptors regulate matrix homeostasis in the course of glomerular injury is therefore critical for devising more effective therapies to treat and ideally prevent glomerulosclerosis.
integrins; collagen; laminin; glomerulus; discoidin domain receptor; fibrosis; growth factors; dystroglycan
Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.
extracellular matrix; fibronectin; integrin; self-renewal; embryonic stem cell
Angiopoietins 1 and 2, ligands for the receptor kinase Tie-2, have been proposed to play critical but opposing roles in vascular development. Since signaling by Tie-2 is likely affected by other endothelial cell receptors such as Flk-1, the receptor for VEGF, and cell-cell adhesion receptors PECAM1 and VE-cad, we explored their interactions in a 3D model of vasculogenesis. When murine embryoid bodies (EBs) were treated with VEGF in Matrigel in the presence or absence of Ang-1 or Ang-2 for eight days, Ang-1 abrogated vascular sprouting for treatments started at days 0 or 3. In contrast, Ang-2 greatly accelerated vascular sprouting compared to untreated EBs. These results were confirmed in a second model system where VEGF treated HUVECs were grown in Matrigel in the presence or absence of Ang-1 or Ang-2. Since vascular sprouting must be precisely controlled in the developing embryo, it is likely that cell-cell adhesion molecules play a role in sensing the density of vascular sprouts. In this respect, we have shown that PECAM1 and CEACAM1 play essential roles in vascular sprouting. We now show that PECAM1 is associated with Tie-2, becomes phosphorylated on its ITIMs, and recruits the inhibitory phosphatases SHP-1 and SHP-2. In addition, PECAM1 is associated with VE-cad and may similarly regulate its signaling via recruitment of SHP-1/2.
Ang-1; Ang-2; PECAM1; CEACAM1; Tie-2. VE-cad; vasculogenesis; angiogenesis
Growth and metastasis of solid tumors requires induction of angiogenesis to ensure the delivery of oxygen, nutrients and growth factors to rapidly dividing transformed cells. Through either mutations, hypoxia generated by cytoreductive therapies, or when a malignancy outgrows its blood supply, tumor cells undergo a change from an avascular to a neovascular phenotype, a transition mediated by the hypoxia-inducible factor (HIF) family of transcriptional regulators. Vascular endothelial growth factor (VEGF) is one example of a gene whose transcription is stimulated by HIF. VEGF plays a crucial role in promoting tumor growth and survival by stimulating new blood vessel growth in response to such stresses as chemotherapy or radiotherapy-induced hypoxia, and it therefore has become a tempting target for neutralizing antibodies in the treatment of advanced neoplasms. Emerging evidence has shown that the semaphorins, proteins originally associated with control of axonal growth and immunity, are regulated by changes in oxygen tension as well and may play a role in tumor-induced angiogenesis. Through the use of RNA interference, in vitro and in vivo angiogenesis assays and tumor xenograft experiments, we demonstrate that expression of semaphorin 4D (SEMA4D), which is under the control of the HIF-family of transcription factors, cooperates with VEGF to promote tumor growth and vascularity in oral squamous cell carcinoma (OSCC). We use blocking antibodies to show that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of OSCC and other solid tumors.
Semaphorin 4D; Plexin-B1; VEGF; oral squamous cell carcinoma; angiogenesis; HIF
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP) -1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.
MLK3; MMP; invasion; ovarian cancer; MAPK; AP-1
The adaptors IRS1 and IRS2 link growth factor receptors to downstream signaling pathways that regulate proliferation and survival. Both suppress factor-withdrawal-induced apoptosis and have been implicated in cancer progression. However, recent studies suggest IRS1 and IRS2 mediate differential functions in cancer pathogenesis. IRS1 promoted breast cancer proliferation, while IRS2 promoted metastasis. The role of IRS1 and IRS2 in controlling cell responses to chemotherapy is unknown. To determine the role of IRS1 and IRS2 in the sensitivity of cells to chemotherapy, we treated 32D cells lacking or expressing IRS proteins with various concentrations of chemotherapeutic agents. We found that expression of IRS1, in contrast to IRS2, enhanced the sensitivity of 32D cells to chemotherapy-induced apoptosis. When IRS2 was expressed with IRS1, the cells no longer showed enhanced sensitivity. Expression of IRS1 did not alter the expression of pro- and anti-apoptotic proteins; however, 32D-IRS1 cells expressed higher levels of Annexin A2. In 32D-IRS1 cells, IRS1 and Annexin A2 were both located in cytoplasmic and membrane fractions. We also found that IRS1 coprecipitated with Annexin A2, while IRS2 did not. Decreasing Annexin A2 levels reduced 32D-IRS1 cell sensitivity to chemotherapy. These results suggest IRS1 enhances sensitivity to chemotherapy in part through Annexin A2.
insulin receptor substrate; signaling; annexin A2; chemotherapy; cell death
Although many human melanomas express the death receptors TRAIL-R2/DR5 or TRAIL-R1/DR4 on cell surface, they often exhibit resistance to exogenous TRAIL. One of the main contributors to TRAIL-resistance of melanoma cells is upregulation of transcription factors STAT3 and NF-κB that control the expression of antiapoptotic genes, including cFLIP and Bcl-xL. On the other hand, the JNK-cJun pathway is involved in the negative regulation of cFLIP (a caspase-8 inhibitor) expression. Our observations indicated that resveratrol, a polyphenolic phytoalexin, decreased STAT3 and NF-κB activation, while activating JNK-cJun that finally suppressed expression of cFLIP and Bcl-xL proteins and increased sensitivity to exogenous TRAIL in DR5-positive melanomas. Interestingly, resveratrol did not increase surface expression of DR5 in human melanomas, while γ-irradiation or sodium arsenite treatment substantially upregulated DR5 expression. Hence, an initial increase in DR5 surface expression (either by γ-irradiation or arsenite), and subsequent downregulation of antiapoptotic cFLIP and Bcl-xL (by resveratrol), appear to constitute an efficient approach to reactivate apoptotic death pathways in TRAIL-resistant human melanomas. In spite of partial suppression of mitochondrial function and the mitochondrial death pathway, melanoma cells still retain the potential to undergo the DR5-mediated, caspase-8-dependent death pathway that could be accelerated by either an increase in DR5 surface expression or suppression of cFLIP. Taken together, these results suggest that resveratrol, in combination with TRAIL, may have a significant efficacy in the treatment of human melanomas.
Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (−)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in expression of NFP, NSE, and e-cadherin and also decreases in expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro network formation ability of cells was significantly inhibited by survivin silencing and completely by combination of survivin silencing and EGCG treatment. Collectively, survivin silencing potentiated anti-cancer effects of EGCG in human malignant neuroblastoma cells having survivin overexpression.
Angiogenesis; Apoptosis; Differentiation; (−)-Epigallocatechin-3-Gallate; Neuroblastoma, Survivin shRNA
Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-JunS73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.
•Sphingosine-1-phosphate induces glioma cell migration and invasion.•Part of the effects is mediated by protein kinase D2 (PRKD2) activation.•Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion.•Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK signaling.•PRKD2 regulates transcription of gene products implicated in migration and invasion.
Glioblastoma; Sphingosine-1-phosphate; PRKD2; MAPK; C-Jun; Invasion