Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells.

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

Differential Expression of Adhesion-Related Proteins and MAPK Pathways Lead to Suitable Osteoblast Differentiation of Human Mesenchymal Stem Cells Subpopulations.

Cellular adhesion enables communication between cells and their environment. Adhesion can be achieved throughout focal adhesions and its components influence osteoblast differentiation of human mesenchymal stem cells (hMSCs). Because cell adhesion and osteoblast differentiation are closely related, this article aimed to analyze the expression profiles of adhesion-related proteins during osteoblastic differentiation of two hMSCs subpopulations (CD105(+) and CD105(-)) and propose a strategy for assembling bone grafts based on its adhesion ability. In vitro experiments of osteogenic differentiation in CD105(-) cells showed superior adhesion efficiency and 2-fold increase of α-actinin expression compared with CD105(+) cells at the maturation stage. Interestingly, levels of activated ß1-integrin increased in CD105(-) cells during the process. Additionally, the CD105(-) subpopulation showed 3-fold increase of phosphorylated FAK(Y397) compared to CD105(+) cells. Results also indicate that ERK1/2 was activated during CD105(-) bone differentiation and participation of mitogen-activated protein kinase (MAPK)-p38 in CD105(+) differentiation through a focal adhesion kinase (FAK)-independent pathway. In vivo trial demonstrated that grafts containing CD105(-) showed osteocytes embedded in a mineralized matrix, promoted adequate graft integration, increased host vascular infiltration, and efficient intramembranous repairing. In contrast, grafts containing CD105(+) showed deficient endochondral ossification and fibrocartilaginous tissue. Based on the expression of α-actinin, FAKy,(397) and ERK1/2 activation, we define maturation stage as critical for bone graft assembling. By in vitro assays, CD105(-) subpopulation showed superior adhesion efficiency compared to CD105(+) cells. Considering in vitro and in vivo assays, this study suggests that integration of a scaffold with CD105(-) subpopulation at the maturation stage represents an attractive strategy for clinical use in orthopedic bioengineering.

Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential.

Human fetal mesenchymal stem cells can be isolated from the amniotic membrane (AM-hMSCs) by enzymatic digestion. The biological properties of this cell population have been characterized; however, few studies have focused on the presence of stem cell subpopulations and their differentiation potential. The aim of the present study was to isolate homogeneous AM-hMSC subpopulations based on the coexpression of surface markers. In addition, we aimed to characterize stem cell subpopulations through the detection of typical stem cell markers and its differentiation potential. In this study, fluorescence-activated cell sorting (FACS) was used to positively select for the surface markers CD44, CD73, and CD105. Two subpopulations were isolated: CD44+ / CD73+ / CD105+ (CD105+), and CD44+ / CD73+ / CD105- (CD105-). To characterize the cell subpopulations, the expression of pluripotency-associated markers was analyzed by reverse transcriptase-polymerase chain reaction and immunofluorescence. Our results showed positive expression of SOX2, SOX3, PAX6, OCT3/4, and NANOG in the CD105+ and CD105(-) cell subpopulations. In contrast, we did not detect expression of SSEA4 or FOXD3 in either subpopulation. Immunophenotypes, such as mesenchymal and hematopoietic markers, were studied by FACS analyses. Our data revealed the expression of the CD49a, CD49d, CD29, integrin α9ß1, CD44, CD73, and CD105 antigens in both subpopulations. In contrast, CD90, CD45, CD34, CD14, and HLA-DR expression was not detected. The ability of both subpopulations to differentiate into osteoblasts, adipocytes, and chondrocytes was evidenced using Alizarin red, Oil-Red, and Alcian blue staining, respectively. Furthermore, neuronal differentiation was demonstrated by the expression of GFAP and NEURO-D. Interestingly, we observed a dissimilar osteoblastic differentiation potential between the subpopulations. CD105- cells showed stronger expression of secreted protein acidic and rich in cysteine (SPARC) and osteonectin, which was associated with more effective calcium deposition, than CD105+ cells. In conclusion, we described a systematic method for the isolation of hMSCs that was highly reproducible and generated homogeneous cultures for osteoblast differentiation with an efficient capacity for mineralization.