Identification and cytoprotective function of a novel nestin isoform, Nes-S, in dorsal root ganglia neurons.

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

MT1-MMP regulates MMP-2 expression and angiogenesis-related functions in human umbilical vein endothelial cells.

Membrane type 1 (MT1)-MMP is a member of matrix metalloproteinases (MMPs) that regulates extracellular matrix remodeling. In addition, MT1-MMP also serves as a multi-functional protein. However, the functional role of MT1-MMP in human endothelial cells remains unclear. In this study we use real-time PCR and Western blotting to demonstrate for the first time that MMP-2 expression is regulated by MT1-MMP in human endothelial cells. Moreover, MMP-2 activity is also modulated by MT1-MMP. In addition we found that endothelial cells, ECM adhesion and human endothelial cell tube formation, which are known to be regulated by MMP-2, are blocked by MT1-MMP siRNA. These results suggest that MT1-MMP plays an important role in regulating angiogenesis in human endothelial cells.

Fibroblast growth factor-2 up-regulates the expression of nestin through the Ras-Raf-ERK-Sp1 signaling axis in C6 glioma cells.

Nestin is a 240-kDa intermediate filament protein expressed mainly in neural and myogenic stem cells. Although a substantial number of studies have focused on the expression of nestin during development of the central nervous system, little is known about the factors that induce and regulate its expression. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and stimulates the proliferation and differentiation of a subset of nestin-expressing cells, including neural progenitor cells, glial precursor cells, and smooth muscle cells. To assess whether FGF-2 is a potent factor that induces the expression of nestin, C6 glioma cells were used. The results showed that nestin expression was up-regulated by FGF-2 via de novo RNA and protein synthesis. Our RT-PCR results showed that C6 glioma cells express FGFR1/3, and FGFRs is required for FGF-2-induced nestin expression. Further signaling analysis also revealed that FGF-2-induced nestin expression is mediated through FGFR-MAPK-ERK signaling axis and the transcriptional factor Sp1. These findings provide new insight into the regulation of nestin in glial system and enable the further studies on the function of nestin in glial cells.

Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration.

Adhesive interactions between cells are needed to maintain tissue architecture during development, tissue renewal and wound healing. Thrombomodulin (TM) is an integral membrane protein that participates in cell-cell adhesion through its extracellular lectin-like domain. However, the molecular basis of TM-mediated cell-cell adhesion is poorly understood. Here, we demonstrate that TM is linked to the actin cytoskeleton via ezrin. In vitro binding assays showed that the TM cytoplasmic domain bound directly to the N-terminal domain of ezrin. Mutational analysis of the TM cytoplasmic domain identified (522)RKK(524) as important ezrin-binding residues. In epidermal epithelial A431 cells, TM colocalized with ezrin and actin filaments at cell-cell contacts. Knockdown of endogenous TM expression by RNA interference induced morphological changes and accelerated cell migration in A431 cells. Moreover, epidermal growth factor, upstream of ezrin activation, stimulated the interaction between ezrin and TM. In skin wound healing of mice, TM and ezrin were highly expressed in neoepidermis, implying that both proteins are key molecules in reepithelialization that requires collective cell migration of epithelial cells. Finally, exogenous expression of TM in TM-deficient melanoma A2058 cells promoted collective cell migration. In summary, TM, which associates with ezrin and actin filaments, maintains epithelial morphology and promotes collective cell migration.

The expression of nestin delineates skeletal muscle differentiation in the developing rat esophagus.

The muscularis externa of the developing rodent esophagus is initially composed of smooth muscle, and later replaced by skeletal muscle in a craniocaudal progression. There is growing evidence of distinct developmental origins for esophageal smooth and skeletal muscles. However, the identification of skeletal muscle progenitor cells is controversial, and the detailed cell lineage of their descendants remains elusive. In the current study, we carried out multiple labeling immunofluorescence microscopy of nestin and muscle type-specific markers to characterize the dynamic process of rat esophageal myogenesis. The results showed that nestin was transiently expressed in immature esophageal smooth muscle cells in early developing stages. After nestin was downregulated in smooth muscle cells, a distinct population of nestin-positive cells emerged as skeletal muscle precursors. They were mitotically active, and subsequently co-expressed MyoD, followed by the embryonic and later the fast type of skeletal muscle myosin heavy chain. Thus, the cell lineage of esophageal skeletal muscle differentiation was established by an immunotyping approach, which revealed that skeletal myocytes arise from a distinct lineage rather than through transdifferentiation of smooth muscle cells during rat esophageal myogenesis.

Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells.

Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.

Epidermal growth factor up-regulates the expression of nestin through the Ras-Raf-ERK signaling axis in rat vascular smooth muscle cells.

The contractile-synthetic phenotypic modulation of vascular smooth muscle cells (VSMCs) is a key event during atherosclerosis progression. Although many studies have reported possible cytokines and growth factors implicated to this process, the critical factors affecting the VSMC phenotype remain unclear due to the lack of early de-differentiation marker identifications. In this study, we showed that nestin, an intermediate filament protein, is expressed in primary cultures of rat VSMCs representing the synthetic phenotype and its expression is diminished as these cells re-differentiate after serum deprivation. However, the regulation of nestin expression was never reported despite its common usage as an early differentiation marker. Herein, we showed that nestin expression is regulated by epidermal growth factor (EGF) via de novo RNA and protein synthesis. Furthermore, signaling analyses revealed that the EGF-induced nestin re-expression is mediated through the activation of the Ras-Raf-ERK signaling axis. This is the first report to show that nestin expression is regulated by an extracellular signaling molecule.

Proliferation of a subpopulation of reactive astrocytes following needle-insertion lesion in rat.

It is well known that traumatic injuries of the CNS induce a gliotic reaction, characterized by the presence of reactive astrocytes. Reactive astrocytes exhibit enhanced expression of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), hypertrophy, and thickened processes. Recently, we have demonstrated that injuries of the CNS induce a re-expression of an embryonic intermediate filament-associated protein, IFAP-70/280 kDa. Based on IFAP-70/280 kDa immunolabeling, we have shown that reactive astrocytes, activated by stab-wound injury, can be divided into two major groups: 1. persistent IFAP+/GFAP+ cells which are close to the wound in the area of glial scar, and 2. transient IFAP-/GFAP+ cells which are farther from the wound. In this study, we use BrdU incorporation to examine proliferation in these two groups of reactive astrocytes induced by stab injury of the rat cerebrum. Triple/double-label immunofluorescence microscopy was performed using antibodies to IFAP-70/280 kDa, GFAP, and BrdU. The results showed that BrdU+ reactive astrocytes (GFAP+) were always IFAB-70/280 kDa+ as well. However, not all IFAP+ reactive astrocytes are BrdU+. BrdU+ signal was not observed in any IFAP- reactive astrocytes. At five days post-lesion, IFAP+ reactive astrocytes were increasing in the area of the wound (0-50 micrograms from the wound edge), but had reached a peak in the proximal area (50-800 micrograms away from the wound edge). At eight days post-lesion, IFAP+ reactive astrocytes achieved the highest percentage in the wound area. At the same time, BrdU-containing reactive astrocytes occupied an area closer to the wound. By 20 days post-lesion, following the formation of the gliotic scar at the stab-wound, a few IFAP+/GFAP+ cells still persisted. BrdU-containing reactive astrocytes were only observed in the scar. These results indicate that many IFAP+ reactive astrocytes close to the wound, in contrast to the IFAP- ones farther from the wound, appear to regain their proliferative potential to increase in number and participate in the formation of the gliotic scar.

Identification of a rod domain-truncated isoform of nestin, Nes-SΔ107₋254, in rat dorsal root ganglia.

Nestin, a type VI intermediate filament (IF) protein, is predominantly expressed in neurogenic and myogenic stem cells. We previously identified the first isoform of nestin, Nes-S, in rat DRG neurons. In this study, we report a previously unidentified nestin isoform, Nes-SΔ107₋254, that is expressed in lower levels in DRG neurons of adult rats. The 29-kD Nes-SΔ107₋254 is identical to Nes-S, except that an additional region is removed in its rod domain. Nes-SΔ107₋254 is assembly compromised and forms aggregates. Unlike nestin and Nes-S, Nes-SΔ107₋254 does not exert cytoprotective effect. Furthermore, elevated caspase-3 activation was observed in HEK293T cells expressing the EGFP-Nes-SΔ107₋254 protein. Taken together, Nes-SΔ107₋254 is a rod domain-truncated isoform of nestin that is susceptible to form cytotoxic aggregates.

Nestin serves as a prosurvival determinant that is linked to the cytoprotective effect of epidermal growth factor in rat vascular smooth muscle cells.

Nestin is an intermediate filament protein mainly expressed in muscle and neural progenitors. Recently, we reported that nestin is expressed in rat vascular smooth muscle cells (VSMCs), disappears after serum-deprivation and then is re-expressed again following EGF stimulation. As the function of nestin in VSMCs remains unknown, its anti-apoptotic function was investigated in this study. We first showed that cell viability of nestin-depleted cells following H(2)O(2) treatments decreased by nestin RNAi. Further DNA laddering analysis and flow cytometry results demonstrated that this loss of cell viability was mediated through apoptosis. In addition, caspase-9, caspase-3 and PARP were activated in nestin-depleted VSMCs following H(2)O(2) treatments, indicating that nestin has an upstream inhibitory effect on caspase activation. It is well known that EGF serves as a survival factor in rat VSMCs. Here, we show that the cytoprotective effect of EGF was prevented by nestin RNAi. In addition, the inhibition of Cdk5 prevented Bcl-2 phosphorylation and enhanced H(2)O(2)-induced caspase-3 activation as well as subsequent DNA fragmentation. Taken together, these results provide evidence for another cytoprotective role of EGF in that it is mediated through its stimulation of nestin expression which leads to the prevention of caspase activation by Cdk-5-induced Bcl-2 phosphorylation in rat VSMCs.

Thrombin induces nestin expression via the transactivation of EGFR signalings in rat vascular smooth muscle cells.

Regulation of nestin gene expression is largely unknown despite that it is widely used as a progenitor cell marker. In this study, we showed that nestin expression is regulated by the thrombin-mediated EGFR transactivation in serum-deprived primary cultures of rat vascular smooth muscle cells (VSMCs). This resulted from the direct binding of thrombin to PAR-1 rather than indirectly affecting through the binding to thrombomodulin, as demonstrated by thrombomodulin RNAi. In this process, the PAR-1-induced c-Src plays a critical role through two routes; one was the direct intracellular phosphorylation of EGFR and the other was the extracellular activation of the MMP-2-mediated shedding of HB-EGF. The transactivated EGFR then led to the downstream Ras-Raf-ERK signaling axis, but not the p38 or JNK pathways. In addition, the EMSA experiment showed that the transcriptional factor Sp1 is critical for the thrombin-induced nestin expression in rat VSMCs. Furthermore, RNAi of nestin attenuated the thrombin-induced cell proliferation, indicating that thrombin-induced nestin expression and cell proliferation share the same EGFR transactivation mechanism. This study also suggested that nestin may play an important role in cell proliferation induced by the thrombin-mediated EGFR transactivation.

Lysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kappaB-dependent mechanism.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G(i), ammonium pyrrolidinedithiocarbamate and BAY 11-7082, inhibitors of the nuclear factor (NF)-kappaB pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G(i)-, NF-kappaB-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes.

Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain.

Thrombomodulin (TM) is an integral membrane glycoprotein that is a potent anticoagulant factor. TM may also possess functions distinct from its anticoagulant activity. Here the influence of TM on cell adhesion was studied in TM-negative melanoma A2058 cells transfected with green fluorescent protein-tagged TM (TMG) or lectin domain-deleted TM (TMG(DeltaL)). Confocal microscopy demonstrated that both TMG and TMG(DeltaL) were distributed in the plasma membrane. TMG-expressed cells grew as closely clustered colonies, with TM localized prominently in the intercellular boundaries. TMG(DeltaL)-expressed cells grew singly. Overexpression of TMG, but not TMG(DeltaL), decreased monolayer permeability in vitro and tumor growth in vivo. The cell-to-cell adhesion in TMG-expressed cells was Ca2+-dependent and was inhibited by monoclonal antibody against the lectin-like domain of TM. The effects of TM-mediated cell adhesion were abolished by the addition of mannose, chondroitin sulfate A, or chondroitin sulfate C. In addition, anti-lectin-like domain antibody disrupted the close clustering of the endogenous TM-expressed keratinocyte HaCaT cell line derived from normal human epidermis. Double-labeling immunofluorescence staining revealed similar distributions of TM and actin filament in the cortex region of the TMG-expressed cells. Thus, TM can function as a Ca2+-dependent cell-to-cell adhesion molecule. Binding of specific carbohydrates to the lectin-like domain is essential for this specific function.