Cytoplasmic-targeted parvalbumin blocks the proliferation of multipotent mesenchymal stromal cells in prophase.
BACKGROUND
Multipotent mesenchymal stromal cells (MSCs) have gained considerable interest because of their potential use in the treatment of a variety of diseases and injuries. Although remarkable advancements have been made in clinical studies, substantial concerns still regard the safety of MSCs. Some evidence suggests that MSCs can spontaneously generate a population of cells with tumorigenic potential. Thus, studying the molecular mechanisms that control the proliferation of MSCs may be a necessary step toward the development of strategies for safe clinical practice. Ca(2+) is a second messenger that mediates a wide range of cellular responses, including the regulation of cell proliferation, but little is known about its function in MSCs. The aim of this study was to investigate the effects of targeted Ca(2+) buffering on MSCs proliferation in vitro.


METHODS
Here, we used an adenoviral (Ad) vector encoding the Ca(2+) chelator protein parvalbumin (PV) fused to a nuclear exclusion signal (NES) and the Discosoma red fluorescent protein (DsRed) to investigate the function of cytoplasmic Ca(2+) signals on MSC proliferation. Confocal microscopy was used to demonstrate that PV-NES-DsRed was expressed in the cytoplasm. Ca(2+) signaling was monitored by using Fluo-4-AM. Fluorescence-activated cell sorting (FACS) analysis of cells that were stained with propidium iodide was used as a quantitative measure of cell death. The mitotic index was assessed by immunofluorescence, and the expression of cyclins was examined with Western blot.


RESULTS
Our results show that the Ad-PV-NES-DsRed fusion protein decreased serum-induced Ca(2+) signaling and blocked the proliferation of rat adipose-derived MSCs (AT-MSCs) in prophase. FACS analysis revealed that Ad-PV-NES-DsRed did not induce cell death in AT-MSCs. Furthermore, Western blot analysis demonstrated that Ad-PV-NES-DsRed reduced extracellular signal-regulated kinase (Erk1/2) phosphorylation and cyclin B1 expression. Buffering cytosolic Ca(2+) did not alter the expression of cyclins A/D1/D2/D3/E and E2.


CONCLUSIONS
Our results show that cytoplasmic Ca(2+) signals are important for AT-MSCs progression beyond prophase because of their effects on Erk phosphorylation and cyclin B1 expression.
Carolina Soares Barros
CS
Melo
Jerusa Araújo Quintão
JA
Arantes Faria
Natássia Caroline Resende
NC
Corrêa
Carolina
C
de Andrade
Juliana Lott
JL
Carvalho
Alfredo M
AM
Goes
Michele A
MA
Rodrigues
Dawidson Assis
DA
Gomes
D000818:Animals; D002118:Calcium; D049109:Cell Proliferation; D008297:Male; D059630:Mesenchymal Stem Cells; D010320:Parvalbumins / Q000378:metabolism*; D010766:Phosphorylation; D011418:Prophase; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction
D010320:Parvalbumins; D002118:Calcium