Effects of IGFBP3 knockdown on human endometrial mesenchymal stromal cells stress-induced senescence.

Insulin-like growth factor binding protein 3 (IGFBP3) is known for its pleiotropic ability to regulate various cellular processes such as proliferation, apoptosis, differentiation etc. It has recently been shown that IGFBP3 is part of the secretome of senescent human endometrial mesenchymal stromal cells (MESCs) (Griukova et al., 2019) that takes part in paracrine propagation of senescence-like phenotype in MESCs (Vassilieva et al., 2020); however, mechanisms of pro-senescent IGFBP3 action in MESCs remain still unexplored. This study is aimed at elucidating the role of IGFBP3 upregulation in senescent MESCs. IGFBP3 knockdown in MESCs committed to H2O2-induced senescence led to partial abrogation of p21/Rb axis, to elevated ERK phosphorylation and to increase in SA-ß-gal activity. Additionally, MESCs derived from various donors were found to demonstrate different IGFBP3 regulation during stress-induced senescence. Obtained results suggest ambiguous role of IGFBP3 in stress-induced senescence of MESCs.

Outcomes of Deferoxamine Action on H2O2-Induced Growth Inhibition and Senescence Progression of Human Endometrial Stem Cells.

Mesenchymal stem cells (MSCs) are broadly applied in regenerative therapy to replace cells that are lost or impaired during disease. The low survival rate of MSCs after transplantation is one of the major limitations heavily influencing the success of the therapy. Unfavorable microenvironments with inflammation and oxidative stress in the damaged regions contribute to MSCs loss. Most of the strategies developed to overcome this obstacle are aimed to prevent stress-induced apoptosis, with little attention paid to senescence-another common stress reaction of MSCs. Here, we proposed the strategy to prevent oxidative stress-induced senescence of human endometrial stem cells (hMESCs) based on deferoxamine (DFO) application. DFO prevented DNA damage and stress-induced senescence of hMESCs, as evidenced by reduced levels of reactive oxygen species, lipofuscin, cyclin D1, decreased SA-ß-Gal activity, and improved mitochondrial function. Additionally, DFO caused accumulation of HIF-1α, which may contribute to the survival of H2O2-treated cells. Importantly, cells that escaped senescence due to DFO preconditioning preserved all the properties of the initial hMESCs. Therefore, once protecting cells from oxidative damage, DFO did not alter further hMESCs functioning. The data obtained may become the important prerequisite for development of a new strategy in regenerative therapy based on MSCs preconditioning using DFO.

Chondrogenic differentiation followed IGFBP3 loss in human endometrial mesenchymal stem cells.

Insulin-like growth factor binding protein 3 (IGFBP3) is a multifunctional protein, able either to stimulate the cell growth or to promote apoptosis. In particular, IGFBP3 plays significant role in propagation of stress-induced senescence in human endometrium-derived mesenchymal stem cells (MESCs) (Vassilieva et al., 2020). We undertook CRISPR/Cas9-mediated IGFBP3 knockout in an effort to decelerate stress-induced senescence in MESCs, but, unexpectedly, IGFBP3-knockout MESCs culture acquired chondrocyte-like features, such as cell condensation and aggregation. We revealed that IGFBP3-knockout MESCs completely lost CD73 and CD90 MESCs positive surface markers, and significantly decreased expression of CD105 and CD146 MESCs positive surface markers. In addition, we found IGFBP3-knockout MESCs aggregates positively stained for Alcian Blue. We also detected expression of collagen type II in IGFBP3-knockout MESCs. The obtained results indicate that MESCs lost stemness after IGFBP3-knockout and underwent differentiation toward chondrogenic lineage. Our findings can enlighten IGFBP3 role in regulation of MESCs chondrogenesis.

Senescence-messaging secretome factors trigger premature senescence in human endometrium-derived stem cells.

Accumulating evidence suggests that the senescence-messaging secretome (SMS) factors released by senescent cells play a key role in cellular senescence and physiological aging. Phenomenon of the senescence induction in human endometrium-derived mesenchymal stem cells (MESCs) in response to SMS factors has not yet been described. In present study, we examine a hypothesis whether the conditioned medium from senescent cells (CM-old) may promote premature senescence of young MESCs. In this case, we assume that SMS factors, containing in CM-old are capable to trigger senescence mechanism in a paracrine manner. A long-term cultivation MESCs in the presence of CM-old caused deceleration of cell proliferation along with emerging senescence phenotype, including increase in both the cell size and SA-ß-Gal activity. The phosphorylation of p53 and MAPKAPK-2, a direct target of p38MAPK, as well as the expression of p21Cip1 and p16Ink4a were increased in CM-old treated cells with senescence developing whereas the Rb phosphorylation was diminished. The senescence progression was accompanied by both enhanced ROS generation and persistent activation of DNA damage response, comprising protein kinase ATM, histone H2A.X, and adapter protein 53BP1. Thus, we suggest that a senescence inducing signal is transmitted through p16/MAPKAPK-2/Rb and DDR-mediated p53/p21/Rb signaling pathways. This study is the first to demonstrate that the SMS factors secreted in conditioned medium of senescent MESCs trigger a paracrine mechanism of premature senescence in young cells.

Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress.

Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition.

Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H(2)O(2)-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H(2)O(2)-treated hMESCs. ATM inhibition allowed H(2)O(2)-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H(2)O(2)-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.

Insulin receptor-related receptor as an extracellular alkali sensor.

The insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH >7.9. The alkali-sensing property of IRR is conserved in frog, mouse, and human. IRR activation is specific, dose-dependent and quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess.

Inhibition of the EGF receptor and ERK1/2 signaling pathways rescues the human epidermoid carcinoma A431 cells from IFNγ-induced apoptosis.

Interferon gamma (IFNγ) has been demonstrated to inhibit tumor growth in vivo as well as proliferation of multiple types of cultured transformed cells. In this study, we showed that IFNγ promoted progressive death in A431 cells, overexpressing EGF receptor (EGFR). Based on the data provided by evaluating cell morphology, MTT assay, FACS analysis, and cleaved caspase-3 staining we concluded that the major cause of IFNγ-induced A431 cell growth inhibition was not cell cycle arrest, but apoptosis. We investigated a role for the EGFR and ERK1/2 MAPK signaling pathways in IFNγ-induced apoptosis of A431 cells. IFNγ-induced cell death was accompanied by both an increase of the ERK1/2 MAPK activation and a simultaneous reduction of the EGFR activation. Activation of ERK1/2 was crucial for IFNγ-induced cell death because MEK1/2 inhibitors, PD0325901 and U0126 efficiently protected cells from apoptosis by suppressing caspase-3 activation. Even though EGFR tyrosine kinase inhibitor AG1478 also rescued A431 cells from IFNγ-induced apoptosis, unlike MEK1/2 inhibitors, it initiated G 1 arrest. Together, these results suggest that sustained inhibition of both EGFR and ERK1/2 leads to significant protection of the cells from IFNγ-induced apoptosis, indicating important roles for the EGFR tyrosine kinase and ERK1/2 MAP-kinases in regulating A431 cell death.