Thapsigargin‐induced Ca<sup>2+</sup> increase inhibits TGFβ1‐mediated Smad2 transcriptional responses via Ca<sup>2+</sup>/calmodulin‐dependent protein kinase II

Ming, Ming; Manzini, Ivan; Le, Weidong; Krieglstein, Kerstin; Spittau, Björn

doi:10.1002/jcb.22843

Cited by 10 publications

(8 citation statements)

References 31 publications

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“…These data show that heme can inhibit both TGFβ production and the signaling cascade downstream of TGFβ, thereby regulating TGFβ responsive genes such as collagen. In addition, TGFβ signaling was markedly inhibited by the ER stress inducer thapsigargin in NIH3T3 and oligodendroglial OLI-neu cell lines ( Alevizopoulos et al, 1997 ; Ming et al, 2010 ). ER stressor thapsigargin inhibited collagen synthesis as well in chick embryo chondrocytes ( Clark et al, 1994 ).…”

Section: Discussionmentioning

confidence: 98%

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

et al. 2018

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Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.

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Section: Discussionmentioning

confidence: 98%

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

et al. 2018

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“…Regarding ER stress-triggered apoptosis downstream, increased Ca 2+ leakage into the cytoplasm mediated by TG activates the TGF-β/Smad3 pathway [60,61], which induces intrinsic apoptosis in the affected cells [62,63]. In addition, activated caspase-3 (a cysteine protease) triggers DNA fragmentation in cells undergoing apoptosis [27,50].…”

Section: Discussionmentioning

confidence: 99%

Erythropoietin Nanobots: Their Feasibility for the Controlled Release of Erythropoietin and Their Neuroprotective Bioequivalence in Central Nervous System Injury

Nguyen

Koo

et al. 2022

Applied Sciences

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Background: Erythropoietin (EPO) plays important roles in neuroprotection in central nervous system injury. Due to the limited therapeutic time window and coexistence of hematopoietic/extrahematopoietic receptors displaying heterogenic and phylogenetic differences, fast, targeted delivery agents, such as nanobots, are needed. To confirm the feasibility of EPO-nanobots (ENBs) as therapeutic tools, the authors evaluated controlled EPO release from ENBs and compared the neuroprotective bioequivalence of these substances after preconditioning sonication. Methods: ENBs were manufactured by a nanospray drying technique with preconditioning sonication. SH-SY5Y neuronal cells were cotreated with thapsigargin and either EPO or ENBs before cell viability, EPO receptor activation, and endoplasmic reticulum stress-related pathway deactivation were determined over 24 h. Results: Preconditioning sonication (50–60 kHz) for 1 h increased the cumulative EPO release from the ENBs (84% versus 25% at 24 h). Between EPO and ENBs at 24 h, both neuronal cell viability (both > 65% versus 15% for thapsigargin alone) and the expression of the proapoptotic/apoptotic biomolecular markers JAK2, PDI, PERK, GRP78, ATF6, CHOP, TGF-β, and caspase-3 were nearly the same or similar. Conclusion: ENBs controlled EPO release in vitro after preconditioning sonication, leading to neuroprotection similar to that of EPO at 24 h.

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“…Although there are multiple reports of calcium signaling regulating SMAD activity through direct phosphorylation by Ca 2+ /calmodulin-dependent protein kinase II (CAMKII), the effect seems highly context dependent. Whereas CAMKII activity promotes SMAD1 transcriptional activity in undifferentiated mesenchymal cells, CAMKII inactivates SMAD2 in fibroblast-like Cos-1 cells [34, 37, 38]. …”

Section: Discussionmentioning

confidence: 99%

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation

et al. 2016

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The tumor microenvironment, primarily composed of myofibroblasts, directly influences the progression of solid tumors. Through secretion of growth factors, extracellular matrix deposition, and contractile mechanotransduction, myofibroblasts, or cancer-associated fibroblasts (CAFs), support angiogenesis and cancer cell invasion and metastasis. The differentiation of fibroblasts to CAFs is primarily induced by TGF-β from cancer cells. To discover agents capable of blocking CAF differentiation, we developed a high content immunofluorescence-based assay to screen repurposed chemical libraries utilizing fibronectin expression as an initial CAF marker. Screening of the Prestwick chemical library and NIH Clinical Collection repurposed drug library, totaling over 1700 compounds, identified cardiac glycosides as particularly potent CAF blocking agents. Cardiac glycosides are traditionally used to regulate intracellular calcium by inhibiting the Na+/K+ ATPase to control cardiac contractility. Herein, we report that multiple cardiac glycoside compounds, including digoxin, are able to inhibit TGF-β-induced fibronectin expression at low nanomolar concentrations without undesirable cell toxicity. We found this inhibition to hold true for multiple fibroblast cell lines. Using real-time qPCR, we determined that digoxin prevented induction of multiple CAF markers. Furthermore, we report that digoxin is able to prevent TGF-β-induced fibroblast contraction of extracellular matrix, a major phenotypic consequence of CAF differentiation. Assessing the mechanism of inhibition, we found digoxin reduced SMAD promoter activity downstream of TGF-β, and we provide data that the effect is through inhibition of its known target, the Na+/K+ ATPase. These findings support a critical role for calcium signaling during CAF differentiation and highlight a novel, repurposable modality for cancer therapy.

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Thapsigargin‐induced Ca²⁺ increase inhibits TGFβ1‐mediated Smad2 transcriptional responses via Ca²⁺/calmodulin‐dependent protein kinase II

Cited by 10 publications

References 31 publications

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

Erythropoietin Nanobots: Their Feasibility for the Controlled Release of Erythropoietin and Their Neuroprotective Bioequivalence in Central Nervous System Injury

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation

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Thapsigargin‐induced Ca2+ increase inhibits TGFβ1‐mediated Smad2 transcriptional responses via Ca2+/calmodulin‐dependent protein kinase II

Cited by 10 publications

References 31 publications

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

Heme Induces Endoplasmic Reticulum Stress (HIER Stress) in Human Aortic Smooth Muscle Cells

Erythropoietin Nanobots: Their Feasibility for the Controlled Release of Erythropoietin and Their Neuroprotective Bioequivalence in Central Nervous System Injury

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation

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Thapsigargin‐induced Ca²⁺ increase inhibits TGFβ1‐mediated Smad2 transcriptional responses via Ca²⁺/calmodulin‐dependent protein kinase II