Because the hyperpolarization-activated cation-selective current I h makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular
matrix ...component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse
hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3â4 days
in vitro , we observed differences in the amplitude and activation rate of I h between neurones in naive and vitronectin-exposed slices (10 μg ml â1 added to serum-free medium), and between neurones in slices derived from wild-type and vitronectin-deficient mice. The potassium
inward rectifier I K(ir) , activated at similar voltages to I h , was not affected by vitronectin. In CA1, differences in I h amplitude primarily reflected changes in maximum conductance ( G max ): a 23.3% increase to 3.18 ± 0.64 nS from 2.58 ± 0.96 nS ( P < 0.05) in vitronectin-exposed neurones, and a 17.9% decrease to 2.24 ± 0.26 nS from 2.73 ± 0.64 nS ( P < 0.05) in neurones from vitronectin-deficient slices. The voltage of one-half maximum activation ( V ½ ) was not significantly affected by vitronectin exposure (â78.1 ± 2.3 mV versus â80.0 ± 4.9 mV in naive neurones; P > 0.05) or vitronectin deficiency (â83.8 ± 3.1 mV versus â82.0 ± 2.9 mV in wild-type neurones; P > 0.05). In CA3 neurones, changes in I h reflected differences in both G max and V ½ : in vitronectin-exposed neurones there was a 35.4% increase in G max to 1.30 ± 0.49 nS from 0.96 ± 0.26 nS ( P < 0.01), and a +3.0 mV shift in V ½ to â89.8 mV from â92.8 mV ( P < 0.05). The time course of I h activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast
component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current
in vitronectin-exposed cells contributing to the acceleration of I h activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered.
We suggest that vitronectin-stimulated increases in I h may potentially affect excitability under pathological conditions.
Neural stem cells (NSCs) hold great promise for glioma therapy due to their inherent tumor-tropic properties, enabling them to deliver therapeutic agents directly to invasive tumor sites. In the ...present study, we visualized and quantitatively analyzed the spatial distribution of tumor-tropic NSCs in a mouse model of orthotopic glioma in order to predict the therapeutic efficacy of a representative NSC-based glioma therapy. U251.eGFP human glioma was established in the brain of athymic mice, followed by stereotactic injection of CM-DiI-labeled human NSCs posterior-lateral to the tumor site. Confocal microscopy, three-dimensional modeling and mathematical algorithms were used to visualize and characterize the spatial distribution of NSCs throughout the tumor. The pattern of NSC distribution showed a gradient with higher densities toward the centroid of the tumor mass. We estimate that NSC-mediated therapy would eradicate 70–90% of the primary tumor mass and the majority of invasive tumor foci. Our method may serve as a model for optimizing the efficacy of NSC-based glioma therapy.
Liver fibrosis is a reversible wound-healing response involving TGFβ1/SMAD activation of hepatic stellate cells (HSCs). It results from excessive deposition of extracellular matrix components and can ...lead to impairment of liver function. Here, we show that vitamin D receptor (VDR) ligands inhibit HSC activation by TGFβ1 and abrogate liver fibrosis, whereas Vdr knockout mice spontaneously develop hepatic fibrosis. Mechanistically, we show that TGFβ1 signaling causes a redistribution of genome-wide VDR-binding sites (VDR cistrome) in HSCs and facilitates VDR binding at SMAD3 profibrotic target genes via TGFβ1-dependent chromatin remodeling. In the presence of VDR ligands, VDR binding to the coregulated genes reduces SMAD3 occupancy at these sites, inhibiting fibrosis. These results reveal an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis and define a role for VDR as an endocrine checkpoint to modulate the wound-healing response in liver. Furthermore, the findings suggest VDR ligands as a potential therapy for liver fibrosis.
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•Vitamin D receptor (VDR) ligands inhibit hepatic stellate cell activation and fibrosis•Vdr knockout mice spontaneously develop liver fibrosis•TGFβ1 signaling shifts the genome-wide binding locations of VDR•VDR antagonizes SMAD3/TGFβ1 activation of profibrotic genes
In the absence of vitamin D receptor (VDR) signaling, SMADs respond to liver injury by activating a set of genes in hepatic stellate cells that cause fibrosis and liver impairment. Paradoxically, SMAD activity also enables repression of fibrosis by making the chromatin around SMAD target sites more accessible to VDR, which, in the presence of ligands, binds and inhibits SMAD activity.
The depletion of stem cell pools and the accumulation of senescent cells in animal tissues are linked to aging. Planarians are invertebrate flatworms and are unusual in that their stem cells, called ...neoblasts, are constantly replacing old and dying cells. By eliminating neoblasts in worms via irradiation, the biological principles of aging are exposed in the absence of wound healing and regeneration, making planaria a powerful tool for aging research.
Pancreatic neuroendocrine tumors (PanNETs) are a rare but clinically important form of pancreatic neoplasia. To explore the genetic basis of PanNETs, we determined the exomic sequences of 10 ...nonfamilial PanNETs and then screened the most commonly mutated genes in 58 additional PanNETs. The most frequently mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN1, which encodes menin, a component of a histone methyltransferase complex, and 43% had mutations in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain-associated protein) and ATRX (α thalassemia/mental retardation syndrome X-linked). Clinically, mutations in the MEN1 and DAXX/ATRX genes were associated with better prognosis. We also found mutations in genes in the mTOR (mammalian target of rapamycin) pathway in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors.
Pancreatic tumors undergo rapid growth and progression, become resistant to chemotherapy, and recur after surgery. We studied the functions of the solute carrier family 39 member 4 (SLC39A4, also ...called ZIP4), which regulates concentrations of intracellular zinc and is increased in pancreatic cancer cells, in cell lines and mice.
We obtained 93 pancreatic cancer specimens (tumor and adjacent nontumor tissues) from patients who underwent surgery and gemcitabine chemotherapy and analyzed them by immunohistochemistry. ZIP4 and/or ITGA3 or ITGB1 were overexpressed or knocked down with short hairpin RNAs in AsPC-1 and MIA PaCa-2 pancreatic cancer cells lines, and in pancreatic cells from KPC and KPC-ZEB1–knockout mice, and pancreatic spheroids were established; cells and spheroids were analyzed by immunoblots, reverse transcription polymerase chain reaction, and liquid chromatography tandem mass spectrometry. We studied transcriptional regulation of ZEB1, ITGA3, ITGB1, JNK, and ENT1 by ZIP4 using chromatin precipitation and luciferase reporter assays. Nude mice were given injections of genetically manipulated AsPC-1 and MIA PaCa-2 cells, and growth of xenograft tumors and metastases was measured.
In pancreatic cancer specimens from patients, increased levels of ZIP4 were associated with shorter survival times. MIA PaCa-2 cells that overexpressed ZIP4 had increased resistance to gemcitabine, 5-fluorouracil, and cisplatin, whereas AsPC-1 cells with ZIP4 knockdown had increased sensitivity to these drugs. In mice, xenograft tumors grown from AsPC-1 cells with ZIP4 knockdown were smaller and more sensitive to gemcitabine. ZIP4 overexpression significantly reduced accumulation of gemcitabine in pancreatic cancer cells, increased growth of xenograft tumors in mice, and increased expression of the integrin subunits ITGA3 and ITGB1; expression levels of ITGA3 and ITGB1 were reduced in cells with ZIP4 knockdown. Pancreatic cancer cells with ITGA3 or ITGB1 knockdown had reduced proliferation and formed smaller tumors in mice, despite overexpression of ZIP4; spheroids established from these cells had increased sensitivity to gemcitabine. We found ZIP4 to activate STAT3 to induce expression of ZEB1, which induced expression of ITGA3 and ITGB1 in KPC cells. Increased ITGA3 and ITGB1 expression and subsequent integrin α3β1 signaling, via c-Jun-N-terminal kinase (JNK), inhibited expression of the gemcitabine transporter ENT1, which reduced gemcitabine uptake by pancreatic cancer cells. ZEB1-knockdown cells had increased sensitivity to gemcitabine.
In studies of pancreatic cancer cell lines and mice, we found that ZIP4 increases expression of the transcription factor ZEB1, which activates expression of ITGA3 and ITGB1. The subsequent increase in integrin α3β1 signaling, via JNK, inhibits expression of the gemcitabine transporter ENT1, so that cells take up smaller amounts of the drug. Activation of this pathway might help mediate resistance of pancreatic tumors to chemotherapeutic agents.
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