The role of lysyl oxidase-like 2 in the odontogenic differentiation of human dental pulp stem cells

Abstract

Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.

Fig. 1.

Morphology and surface marker expression of hDPSCs. (A) The hDPSCs that grew from the dental pulp showed a fibroblast-like morphology. Magnification, 40x. (B) A representative results of FACS analysis of the cell surface protein markers CD44, CD73 and CD105 (MSC surface markers), and CD34 (hematopoietic surface protein) as a negative marker. (C) Confirmation of the ability of hDPSCs to differentiate into odontogenic/osteogenic cells via analysis of mRNA expression levels of alkaline phosphatase (ALP). ^*P < 0.05 versus control.

Fig. 2.

Confirmation of LOXL2 expressions in early odontogenic differentiation. (A) The lysyl oxidase-like 2 (LOXL2) mRNA expression levels were confirmed by real-time qRT-PCR for the RNAs isolated from the hDPSCs exposed to either normal growth media (GM) or mineralization media (MM) for 3 days. (B, C) LOXL2 expression levels in total cell lysates (TCL) (B) and in the secreted protein component from conditioned medium (CM) (C), at the early odontogenic induction stage, were determined by Western blot analysis using an anti-LOXL2 antibody. (D) PNGase F treatment of secreted proteins resulted in a smaller size for LOXL2, indicating that its higher molecular weight is partially attributed to N-glycosylation modification. ^*P < 0.05 versus control.

Fig. 3.

The mRNA expression levels and protein levels of LOXL2 during odontogenic differentiation. (A) LOXL2 mRNA expression levels were monitored by qRT-PCR during odontogenic differentiation by mineralization media (MM). (B) The total cell lysates (TCL) of hDPSCs cultured in normal growth media (GM) or mineralization media (MM) for given days subjected to Western blot analysis. (C) The expression levels of LOX family members in the hDPSCs for 3 days of GM or MM were analyzed by real-time qRT-PCR. (D) Shown are magnified subsets of the LOXL3 and LOXL4 data from (C). ^*P < 0.05 versus control.

Fig. 4.

The effect of rhLOXL2 treatment on hDPSCs. (A, B) hDPSCs were treated with normal growth media (GM) or mineralization media (MM) containing 10 ng/ml of rhLOXL2 for 7 days and then stained with ALP (A) and Alizarin Red S (B). (C) ALP expression levels were examined by qRT-PCR in hDPSC cells treated with rhLOXL2 at the concentrations of 0.1, 1, and 10 ng/ml in MM for 7 days. (D) The mRNA expression levels of DMP-1, an odontogenic-selective gene, were examined by qRT-PCR in the cells treated with rhLOXL2 at the concentrations of 10, 50, and 100 ng/mL in MM for 7 days, (E) DSPP, another onodogenic selective gene, expression levels were also examined by qRT-PCR in hDPSCs treated with rhLOXL2 at concentrations of 1, 10 ng/ml in MM for 7 days. ^*,^#p < 0.05 versus control.