Activation of p70S6 Kinase-1 in Mesenchymal Stem Cells Is Essential to Lung Tissue Repair

Abstract

All-trans retinoic acid (ATRA) or mesenchymal stem cells (MSCs) have been shown to promote lung tissue regeneration in animal models of emphysema. However, the reparative effects of the combination of the two and the role of p70S6 kinase-1 (p70S6k1) activation in the repair process have not been defined. Twenty-one days after intratracheal instillation of porcine pancreatic elastase (PPE), MSC and/or 10 days of ATRA treatment was initiated. Thirty-two days later, static lung compliance (Cst), mean linear intercepts (MLIs), and alveolar surface area (S) were measured. After PPE, mice demonstrated increased values of Cst and MLI, and decreased S values. Both ATRA and MSC transfer were individually effective in improving these outcomes while the combination of ATRA and MSCs was even more effective. The combination of p70S6k1^-/- MSCs transfer followed by ATRA demonstrated only modest effects, and rapamycin treatment of recipients with wild-type (WT) MSCs and ATRA failed to show any effect. However, transfer of p70S6k1 over-expressing-MSCs together with ATRA resulted in further improvements over those seen following WT MSCs together with ATRA. ATRA activated p70S6k1 in MSCs in vitro, which was completely inhibited by rapamycin. Tracking of transferred MSCs following ATRA revealed enhanced accumulation and extended survival of MSCs in recipient lungs following PPE but not vehicle instillation. These data suggest that in MSCs, p70S6k1 activation plays a critical role in ATRA-enhanced lung tissue repair, mediated in part by prolonged survival of transferred MSCs. p70S6k1-activated MSCs may represent a novel therapeutic approach to reverse the lung damage seen in emphysema. Stem Cells Translational Medicine 2018;7:551-558.

Keywords: Animal models; Cell signaling; Cellular therapy; Mesenchymal stem cells; Tissue regeneration.

Figure 1

The reparative effects of combination treatment with MSC and ATRA on lung tissue in elastase‐induced emphysema. The treatment groups were: (1) vehicle alone, (2) MSCs, (3) ATRA administration for 10 days, or (4) MSC/ATRA initiated 21 days after elastase (PPE) or saline (SAL) installation (IT). Twelve days after the initiation of the treatment, airway function and lung tissue sampling were performed. (A): Representative lung tissue sections (H&E staining) from mice which received: a. intratracheal vehicle instillation, b. elastase instillation followed by vehicle treatment, c. elastase installation followed by ATRA treatment for 10 days, d. elastase installation followed by MSC transfer, and e. elastase installation followed by MSC/ATRA treatment. Values of MLI (B), alveolar surface area (S) (C) and static compliance (Cst) (D) following MSC or/and ATRA treatments. n = 8 in each group. *, p < .05 versus vehicle‐PPE, +, p < .05 versus ATRA/PPE and MSC/PPE. Abbreviations: ATRA, all‐trans retinoic acid; MLI, mean linear intercept; MSC, mesenchymal stem cell; PPE, porcine pancreatic elastase.

Figure 2

The role of p70S6k1 phosphorylation in MSC/ATRA‐dependent lung tissue repair. Treatment with vehicle alone, transfer of WT‐MSCs, or p70S6k1‐deficient (S6k1^−/−) MSCs with ATRA administration for 10 days was initiated 21 days after elastase (PPE) or saline (SAL) installation (IT). Twelve days after the initiation of the treatment, airway function and lung tissue sampling were performed. (A): Representative lung tissue sections (H&E staining) from mice which received: a. intratracheal vehicle instillation, b. elastase instillation followed by vehicle treatment, c. elastase instillation followed by treatment with WT‐MSCs transfer and ATRA administration, and d. elastase instillation followed by transfer of S6k1^−/− MSCs and ATRA administration. Values of mean linear intercept (MLI) (B), alveolar surface area (S) (C) and static compliance (Cst) (D) following PBS as vehicle, or WT‐MSCs, or S6k1^−/− MSCs with ATRA treatments. MLI (E), alveolar surface area (S) (F) and Cst (G) values from mice treated with rapamycin or vehicle in parallel with WT‐MSC/ATRA treatment following elastase installation. (H): Immunoblot analysis of phosphorylated p70S6k1 levels (ppS6k1) in MSCs, which were cocultured with ATRA or ATRA following rapamycin pretreatment. n = 8 in each group. *, p < .05 versus PBS/vehicle‐PPE, +, p < .05 versus S6k1^−/− MSC/ATRA‐PPE or rapamycin/ATRA/WT‐MSC‐PPE. Abbreviations: ATRA, all‐trans retinoic acid; MLI, mean linear intercept; MSC, mesenchymal stem cell; PBS, phosphate buffered saline; PPE, porcine pancreatic elastase; WT, wild‐type.

Figure 3

The effects of p70S6k1 over‐expressing MSCs on MSC/ATRA‐dependent lung tissue repair. (A): Immunoblot analysis of whole protein levels of p70S6k1 in MSCs following lentiviral transfection. Values of MLI (B), alveolar surface area (S) (C), and Cst (D) in mice that received transfer of WT‐MSCs or p70S6k1 over‐expressing (S6k1OE) MSCs with ATRA administration following elastase (PPE) or saline (SAL) instillation (IT). n = 8 in each group. *, p < .05 versus PBS/vehicle‐PPE, +, p < .05 versus saline. Abbreviations: ATRA, all‐trans retinoic acid; MLI, mean linear intercept; MSCs, mesenchymal stem cells; PBS, phosphate buffered saline; PPE, porcine pancreatic elastase; WT, wild‐type.

Figure 4

Increased numbers of labeled MSCs in lungs following transfer. Numbers of transferred tdTomato⁺ MSCs in lungs were analyzed by flow cytometry. Recipient mice received elastase instillation 21 days prior to MSC transfer, and ATRA (PPE + MSC/ATRA) or vehicle (PPE + MSC/vehicle) were administered following the transfer until the last experimental days. A group of mice received saline instillation 21 days prior to MSC transfer and ATRA treatment (saline + MSC/ATRA). (A): Ratio of tdTomato⁺ cells to total lung cells. (B): Estimated absolute numbers of tdTomato⁺ cells per lung. n = 6 in each group. *, p < .05 versus PPE + MSC/vehicle and saline + MSC/ATRA. Abbreviations: ATRA, all‐trans retinoic acid; MSCs, mesenchymal stem cells; PPE, porcine pancreatic elastase.

Figure 5

Proposed mechanisms of MSC/ATRA‐dependent repair effects on damaged lung tissue in emphysema. ATRA resulted in the activation of p70S6k1 in exogenous MSCs and increased accumulation and survival time in the lung, promoting lung tissue repair/regeneration. Abbreviations: ATRA, all‐trans retinoic acid; MSC, mesenchymal stem cell; mTOR, mammalian target of rapamycin.