Id1 Expression Level Determines the Differentiation of Human Dental Pulp Stem Cells

Abstract

TWIST1 plays a crucial role in dentinogenesis, and its activity depends on both a dimerization partner selection and phosphorylation. Other factors, like Id proteins, can affect the availability of dimerization partners for TWIST1, subsequently leading to diverse biological outcomes. The purpose of this study was to evaluate an impact of Id1 expression on differentiation of dental pulp stem cells (DPSCs). The altered expression of Id1 was achieved by transfection of human DPSCs with lentiviral vectors either driving an entire sequence of Id1, hence leading to Id1 overexpression, or carrying the Id1 silencing sequence. We observed that both overexpression and silencing of Id1 modulated human DPSC differentiation. Id1 overexpression resulted in a prevailing formation of TWIST1 homodimer and increased expression of genes encoding dentin sialophosphoprotein and dentin matrix protein 1, which confirm an enhanced odontogenic differentiation of DPSCs. Concurrently, Id1 silencing produced an opposite effect, slowing DPSC differentiation. These results highlight Id1 as an important modulator of molecular events during DPSC commitment and differentiation, which should be considered in dental research on tissue engineering. Moreover, we assume that the balance between TWIST1 dimerization forms in DPSCs might function in a cell-type-specific manner.

Keywords: basic-helix-loop helix proteins; cell differentiation; dentin; dentin sialophosphoprotein; odontoblast; protein dimmerization.

Figure 1.

Lentivirus-mediated expression of Id1 in human DPSCs. (A) DPSCs transfected with the pWPI-GFP-Id1 lentiviral vector (Id1, above) and in DPS cells transfected with the pLL3.7-shId1 vector carrying the Id1 silencing sequences (shId1) visible in normal light (upper panels) and under a fluorescent microscope (lower panel). (B) Quantitative real-time polymerase chain reaction analysis of Id1 transcript levels in DPSCs. Data were normalized to GAPDH mRNA levels and are presented as fold-change relative to control DPSCs transfected with an empty vector or vector carrying non-targeting shRNA. The data represent the mean values ± SD (n = 5). *p < .05 vs. control. (C) The Id1 protein level in DSPCs. The proteins in the cell extract were separated by SDS-PAGE and immunoblotted with appropriate antibodies. β-actin was used as a reference protein. The blots presented are representative of those obtained in 4 experiments performed on unique cell cultures derived from four different patients.

Figure 2.

Impact of altered expression of Id1 on dentin- and bone-related markers of human DPSC differentiation. (A) Quantitative real-time PCR analysis of TWIST1, OC, DSPP, DMP1, and OSF2 transcript levels in DPSCs transfected with the pWPI-GFP-Id1 lentiviral vector (Id1, above) and in DPSCs transfected with the vector carrying the Id1 silencing sequences (shId1). Total RNA was isolated from cells cultured for 14 days after transfection. Data are normalized to GAPDH mRNA level and are presented as fold-change relative to control DPSCs transfected with an empty vector or a vector carrying non-targeting shRNA. Data represent the mean values ± SD (n = 4). *p < .05 vs. control. (B) The TWIST1, OC, DSPP, DMP1, and OSF2 protein levels in DPSC with altered expression of Id1. The proteins in the cell extract (from cells cultured for 14 days after transfection) were separated by SDS-PAGE and immunoblotted with appropriate antibodies. β-actin was used as a reference protein. The blots presented are representative of those obtained in 5 experiments performed on unique cell cultures derived from five different patients.

Figure 3.

Id1 overexpression-induced differentiation of human DPSCs. DPSCs were transfected with the pWPI-GFP-Id1 lentiviral vector (Id1, above) or pLL3.7-shId1 vector carrying the Id1 silencing sequences (shId1). The control DPSCs were transfected with an empty vector or vector carrying non-targeting shRNA. The cells were cultured in DMEM for 14 days after transfection. The cell viability was 90% or greater, as determined by Trypan Blue dye exclusion. (A) The activity of alkaline phosphatase (ALP) in DPSC extracts was assayed by a colorimetric method. The data are the mean values ± SD (n = 5). *p < .05 vs. control. (B) Transcript levels of genes encoding osteopontin (OPN), bone sialoprotein (BSP), osteonectin (ON), and α chain of collagen 1 (COL1A1). Data were normalized to GAPDH mRNA level and are presented as mean fold-change relative to control (0 time) ± SD (n = 3). *p < .05 vs. control. (C) Calcium content in DPSCs with altered expression of Id1. Data are presented as mean values ± SD (n = 3). *p < .05 vs. control. (D) A representative (n = 3) photograph of von Kossa staining of DPSCs cultured in a medium supplemented with 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 µg/mL ascorbic acid. Mineral deposits (white arrow) are visible exclusively in DPSCs overexpressing Id1 (Id1, above), while control cells and DPSCs with silenced Id1 (shId1) are void of mineral deposits. (E) Cell number after 14 days in culture. The data are the mean values ± SD (n = 5). *p < .05 vs. control.

Figure 4.

Impact of Id1 expression status on TWIST1/E12 complex formation in DPSCs. DPSCs were transfected with the pWPI-GFP-Id1 lentiviral vector (Id1, above) or pLL3.7-shId1 vector carrying the Id1 silencing sequences (shId1). The control DPSCs were transfected with an empty vector or vector carrying non-targeting shRNA. The presence of the TWIST1/E12 complex in DPSC protein extracts was determined by the co-immunoprecipitation method with the Pierce Direct IP Kit (Thermo Scientific, Waltham, MA, USA). Agarose beads were coupled to anti-TWIST1 polyclonal antibody. The proteins eluted from agarose were electrophoretically separated and immunoblotted with anti-E12 monoclonal antibody (upper panel). The equivalence of loaded proteins on agarose beads was controlled by β-actin immunoblotting performed on cell extract (lower panel). The blot presented is representative of those obtained in 4 experiments performed on unique cell cultures derived from four different patients.