REAC technology and hyaluron synthase 2, an interesting network to slow down stem cell senescence

Abstract

Hyaluronic acid (HA) plays a fundamental role in cell polarity and hydrodynamic processes, affording significant modulation of proliferation, migration, morphogenesis and senescence, with deep implication in the ability of stem cells to execute their differentiating plans. The Radio Electric Asymmetric Conveyer (REAC) technology is aimed to optimize the ions fluxes at the molecular level in order to optimize the molecular mechanisms driving cellular asymmetry and polarization. Here, we show that treatment with 4-methylumbelliferone (4-MU), a potent repressor of type 2 HA synthase and endogenous HA synthesis, dramatically antagonized the ability of REAC to recover the gene and protein expression of Bmi1, Oct4, Sox2, and Nanog in ADhMSCs that had been made senescent by prolonged culture up to the 30(th) passage. In senescent ADhMSCs, 4-MU also counteracted the REAC ability to rescue the gene expression of TERT, and the associated resumption of telomerase activity. Hence, the anti-senescence action of REAC is largely dependent upon the availability of endogenous HA synthesis. Endogenous HA and HA-binding proteins with REAC technology create an interesting network that acts on the modulation of cell polarity and intracellular environment. This suggests that REAC technology is effective on an intracellular niche level of stem cell regulation.

Figure 1. HAS2 inhibition counteracts REAC-mediated decrease in-SA-Gal staining.

At the indicated passages, confluent (80%) ADhMSCs were exposed for 12 hours in the absence (Control) or presence of REAC (REAC treated), or they were subjected to a 12-hour REAC treatment in the presence of 1 mM 4-MU (HAS2 inhibitor) (REAC treated + 4-MU). Panel A, shows representative (six separate experiments) SA-β-Gal staining (blue color). Panel B, reports the percentage analysis of positively stained cells under each experimental condition (mean ± S.E.; n = 6; P < 0.05).

Figure 2. HAS2 inhibition antagonizes REAC-mediated action on Bmi1 gene expression.

At each indicated passage, ADhMSCs were left untreated (control) or treated for 12 hours with REAC, with or without 1 mM 4-MU. The mRNA level of Bmi1 was normalized to HPRT1 and was expressed as fold change relative to mRNA level at time 0 (untreated cells at passage 5), defined as 1. At each time point, mRNA levels from REAC-treated ADhMSCs were significantly different from those detected in control untreated cells. mRNA levels from cells that had been treated with REAC in the presence of 4-MU were significantly different from the expression levels detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P < 0.05).

Figure 3. HAS2 inhibition blunts the effect of REAC treatment on the transcription of stemness related genes.

At each indicated passage, ADhMSCs were left untreated (control) or subjected to REAC treatment for 12 hours, in the absence or presence of 1 mM 4-MU. The mRNA levels of Oct4 (A), Sox2 (B), or Nanog (C) were normalized to HPRT1 and were expressed as fold of change relative to mRNA level at time 0 (unexposed cells at passage 5), defined as 1. At each time point, mRNA levels from REAC-treated ADhMSCs were significantly different from those detected in control untreated cells. mRNA levels from cells that had been treated with REAC in the presence of 4-MU were significantly different from the expression levels detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P < 0.05).

Figure 4. REAC-mediated rescue of Oct4 and Sox2 and NANOG protein expression is antagonized by HAS2 inhibitor.

Total cellular extracts were obtained from ADhMSCs that had been exposed for 12 hours in the absence (−R) or presence of REAC (+R), or from cells that had been subjected to a 12-hour REAC treatment in the presence of 1 mM 4-MU (+R+I). Total lysate from human iPS was used as a control (iPS). Western blot analyses were performed by the aid of polyclonal antibody directed against the indicated target proteins. Representative of six separate experiments.

Figure 5. HAS2 inhibitor suppresses REAC-mediated recovery of TERT gene expression.

At each indicated passage, ADhMSCs were left untreated (control) or subjected to REAC treatment for 12 hours, in the absence or presence of 1 mM 4-MU. TERT mRNA level was normalized to HPRT1 and was expressed as fold change relative to mRNA level at time 0 (unexposed cells at passage 5), defined as 1. At each time point, mRNA levels from REAC-treated ADhMSCs were significantly different from those detected in control untreated cells. mRNA levels from cells that had been treated with REAC in the presence of 4-MU were significantly different from the expression levels detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P < 0.05).

Figure 6. Telomerase activity.

TRAPEZE-RT assay was performed in control untreated cells or ADhMSCs that had been treated with REAC for 12 h at the indicated passages in the absence or presence of 1 mM 4-MU. All data from REAC-treated cells at each time point were significantly different from those in control untreated cells. Telomerase activity from cells that had been REAC treated in the presence of 4-MU were significantly different from the activity detected in REAC-treated cells in the absence of the HAS2 inhibitor. (mean ± S.E.; n = 6; P < 0.05).