Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks

Noradrenaline Sensitivity Is Severely Impaired in Immortalized Adipose-Derived Mesenchymal Stem Cell Line

Primary adipose tissue-derived multipotent stem/stromal cells (adMSCs) demonstrate unusual signaling regulatory mechanisms, i.e., increased of sensitivity to catecholamines in response to noradrenaline. This phenomenon is called “heterologous sensitization”, and was previously found only in embryonic cells. Since further elucidation of the molecular mechanisms that are responsible for such sensitization in primary adMSCs was difficult due to the high heterogeneity in adrenergic receptor expression, we employed immortalized adipose-derived mesenchymal stem cell lines (hTERT-MSCs). Using flow cytometry and immunofluorescence microscopy, we demonstrated that the proportion of cells expressing adrenergic receptor isoforms does not differ significantly in hTERT-MSCs cells compared to the primary adMSCs culture. However, using analysis of Ca2+-mobilization in single cells, we found that these cells did not demonstrate the sensitization seen in primary adMSCs. Consistently, these cells did not activate cAMP synthesis in response to noradrenaline. These data indicate that immortalized adipose-derived mesenchymal stem cell lines demonstrated impaired ability to respond to noradrenaline compared to primary adMSCs. These data draw attention to the usage of immortalized cells for MSCs-based regenerative medicine, especially in the field of pharmacology

Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor

Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS). Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs). We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII) receptor type 1 (AT1). Using the analysis of Ca2+ mobilization in single cells we found that only 5.2 ± 2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2), which was responsible for increased adipose competency of this ADSC subpopulation

Data supporting that adipose-derived mesenchymal stem/stromal cells express angiotensin II receptors in situ and in vitro

This article contains results of analyses of angiotensin II receptors expression in human adipose tissue and stem/stromal cells isolated from adipose tissue. We also provide here data regarding the effect of angiotensin II on intracellular calcium mobilization in adipose tissue derived stem/stromal cells (ADSCs). Discussion of the data can be found in (Sysoeva et al., 2017) [1]

Nox4 and duox1/2 mediate redox activation of mesenchymal cell migration by PDGF

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt

Expression profile and silencing of NADPH-oxidases in mesenchymal cells.

<p>(<b>A</b>)–(<b>B</b>), RT-PCR of NADPH-oxidases in 3T3 fibroblasts and MSC, respectively. Nox5 was not assessed in 3T3 fibroblasts, because it is absent in these cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154157#pone.0154157.ref030" target="_blank">30</a>]. (<b>C</b>)–(<b>D</b>), 3T3 fibroblasts were stably infected by shRNAs to Nox4 or Duox1 and analyzed for corresponding mRNA (<b>C</b>) and protein expression (<b>D</b>). The graph shows mRNA expression levels normalized to those in cells expressing scrambled shRNA; (*) p < 0.05 as compared to scrambled controls in 3 independent experiments. (<b>E</b>)–(<b>F</b>), 3T3 fibroblasts were transiently transfected by siRNAs to Duox1 or Duox2 and analyzed for expression of mRNA in 3 independent experiments (<b>E</b>) and Nox4 and Duox1/2 proteins in 2 experiments (<b>F</b>). The mRNA expression levels were normalized to those in cells treated with non-targeting (NT) siRNA; (*) p < 0.05 as compared to NT controls. The western blots are typical of 2 experiments. (<b>G</b>)–(<b>H</b>), MSC were transiently transfected by siRNAs to Nox4, Duox1 or Duox2, and analyzed for mRNA in 3 independent experiments (<b>G</b>), and Nox4 protein expression in 2 experiments (<b>H</b>). The mRNA expression levels were normalized to those in NT controls; (*) p < 0.05 as compared to the NT controls. In this case Duox1/2 protein expression was not significantly altered by corresponding siRNAs (data not shown).</p

PDGF stimulates redox-sensitive phosphorylation of PKB/Akt in mesenchymal cells, whereas EGF has no effect.

<p>(<b>A</b>)–(<b>B</b>), representative western blots showing the effects of apocynin (<b>A</b>) or PEG-catalase (<b>B</b>) on phosphorylation kinetics of PKB/Akt and Erk1/2 in PDGF-stimulated 3T3 fibroblasts, and vinculin staining in the same lysates used for the loading control. (<b>C</b>)–(<b>D</b>), the corresponding changes in phosphorylation of PKB/Akt or Erk1/2 in fibroblasts analyzed in 4 independent experiments by normalization of phosphorylation signals exemplified above to the vinculin content. Additionally, each data set was normalized to the value of 10 min stimulation in uninhibited control, which therefore has no error bar; (*) p < 0.05 as compared to uninhibited controls. (<b>E</b>)–(<b>F</b>), representative western blots showing the effects of apocynin (<b>E</b>) or PEG-catalase (<b>F</b>) on phosphorylation kinetics of PKB/Akt and Erk1/2 in PDGF-stimulated MSC, and vinculin staining in the same lysates used for the loading control. (<b>G</b>)–(<b>H</b>), the corresponding changes in phosphorylation of PKB/Akt or Erk1/2 in MSC analyzed in 4 independent experiments by normalization of phosphorylation signals exemplified above to the vinculin content. As above, each data set was normalized to the value of 10 min stimulation in uninhibited control; (*) p < 0.05 as compared to uninhibited controls. (<b>I</b>)–(<b>J</b>), PDGF, but not EGF stimulates phosphorylation of PKB/Akt in 3T3 fibroblasts (<b>I</b>) and MSC (<b>J</b>), but both PDGF and EGF similarly stimulate phosphorylation of Erk1/2. Shown are representative membranes from 2 independent experiments.</p

PDGF, but not EGF stimulates accumulation of H₂O₂ in fibroblasts.

<p>NIH-3T3 fibroblasts were transiently transfected with the plasmid encoding HyPer-NES, the ratiometric cytoplasmic sensor for H₂O₂. (<b>A</b>) and (<b>B</b>) show typical confocal images of the control and apocynin pre-treated cells, respectively, taken at the indicated time points after stimulation with PDGF. The <i>upper</i> and <i>middle</i> image rows show the changes in fluorescence intensity excited by either 405 nm (top) or 488 nm (middle) lasers. The lower row represents ratio images that report relative changes in cytoplasmic H₂O₂. PEG-catalase (40 units/ml) was added 30 min after PDGF to decompose H₂O₂ and detect the baseline of the HyPer fluorescense ratio in cytoplasm. Scale bar, 10 μm. (<b>C</b>), the time course of H₂O₂ accumulation in the control (<i>grey</i>) and apocynin treated (<i>black</i>) cells. Shown are mean values of the HyPer fluorescence ratio ± SE obtained from 38 control and 65 apocynin-treated cells in 4 independent experiments; (*) p < 0.05 as compared to untreated controls. (<b>D</b>)-(<b>Е</b>), 3T3 fibroblasts were consequently treated with 20 ng/ml EGF and 10 ng/ml PDGF to directly compare the H₂O₂ responses. (<b>D</b>) shows confocal images taken at the indicated time points after EGF and PDGF addition depicted underneath. Scale bar, 10 μm. (<b>E</b>) shows the time course of H₂O₂ accumulation in cytoplasm; addition of growth factor is indicated by arrows. Shown are the mean values of HyPer ratio ± SE from 43 cells analyzed in 3 independent experiments.</p