Procaine Inhibits Osteo/Odontogenesis through Wnt/β-Catenin Inactivation

Introduction Periodontitis is a complex pathology characterized by the loss of alveolar bone. The causes and the mechanisms that promote this bone resorption still remain unknown. The knowledge of the critical regulators involved in the alteration of alveolar bone homeostasis is of great importance for developing molecular therapies. Procaine is an anesthetic drug with demethylant properties, mainly used by dentists in oral surgeries. The inhibitor role of Wnt signaling of procaine was described in vitro in colon cancer cells. Methods In this work we evaluated the role of procaine (1 uM) in osteo/odontogenesis of rat bone marrow mesenchymal stem cells. Similarly, the mechanisms whereby procaine achieves these effects were also studied. Results Procaine administration led to a drastic decrease of calcium content, alkaline phosphatase activity, alizarin red staining and an increase in the expression of Matrix Gla Protein. With respect to osteo/odontogenic markers, procaine decreased early and mature osteo/odontogenic markers. In parallel, procaine inhibited canonical Wnt/β-catenin pathway, observing a loss of nuclear β-catenin, a decrease in Lrp5 and Frizzled 3, a significant increase of sclerostin and Gsk3β and an increase of phosphorylated β-catenin. The combination of osteo/ odontogenic stimuli and Lithium Chloride decreased mRNA expression of Gsk3β, recovered by Procaine. Furthermore it was proved that Procaine alone dose dependently increases the expression of Gsk3β and β-catenin phosphorylation. These effects of procaine were also observed on mature osteoblast. Interestingly, at this concentration of procaine no demethylant effects were observed. PLO

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

Procaine prevents osteo/odontogenic differentiation of mesenchymal stem cells (MSC).

<p>Changes in the mRNA expression of A) early markers of oste/odontogenesis and B) specific genes of mesenchymal stem cells (MSC) were determined by RT-PCR after osteo/odontogenic differentiation (OB), OB with procaine (1 μM) (OB+Proc) during 21 days. The size and intensity of amplicon was electrophoresed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping. C) Runx2 transcription factor activity was determined by a commercial TransAM^™ assay. Only OB cells showed to be significantly positive for this transcription factor (*p<0.05 vs all groups). D) Western blot for cytoplasmatic protein smooth muscle-22 alpha after 21 days of osteo/odontogenic differentiation. Images are representative of three cultures.</p

Procaine impairs osteogenic maturation of mesenchymal stem cells (MSC).

<p>mRNA expression of mature markers of oste/odontoblast such as A) DMP1 and B) RANKL were measured by RT-PCR after 21 days in culture. The expression in mesenchymal stem cell differentiated into osteo/odontoblasts (OB) was significantly higher than in undifferentiated cells (UC). This expression was reduced by the addition of Procaine (OB+Proc) during the differentiation process. Ribosomal 18S expression was used as housekeeping (* p<0.05 vs all groups).</p

Mechanism of action of procaine.

<p>Changes in mRNA expression of GSK3β from undifferentiated mesenchymal stem cells (UC), MSC differentiated into osteo/odontoblast (OB), MSC differentiated into osteo/odontoblast plus procaine (1μM) (OB+Proc), MSC differentiated into osteo/odontoblast plus lithium chloride (10 mM) (OB+LiCl) and the combination of osteo/odontogenic stimuli, Procaine (1μM) and Lithium Chloride (10 mM) were analyzed A). Effect of increasing concentrations of Procaine (0, 0.5, 1 and 2 μM) on GSK3βexpression B) and phospho-β-catenin C) for 7 days on MSC. * p<0.05 vs UC; # p<0.05 vs OB; + p<0.01 vs OB+LiCl; ••p<0.01 vs. 0.5 μM Proc.</p

Procaine down-regulates Wnt/β- catenin pathway during osteo/odontogenesis of MSC.

<p>Osteo/odontoblast stimulus (OB) increased the mRNA expression of A) Lrp5/6 and B) Frizzled 1 and decreased the mRNA expression of C) Gsk3β (***p<0.05 vs. undifferentiated cells, UC. Treatments with procaine (OB+Proc) decreased expression of Lrp5/6 and Frizzled 1 and increased expression of Gsk3β (### p<0.001 vs OB cells and ## p<0.01 vs OB cells). D) Rat mesenchymal stem cells treated with osteo/odontoblasts stimulus (OB) or osteo/odontoblasts stimulus plus procaine (OB+Proc) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Original magnification: 40x. Images were representatives of three independent experiments. E) phospho-β-catenin analysis by western blot showed that osteo/odontogenesis of MSC leads to a decrease of this protein while the presence of Procaine recovered the cytoplasmic levels of phospho β-catenin after 21 days of culture. Images are representative of three cultures. F) mRNA expression of sclerostin was decreased by osteo/odontoblast stimulus (OB) (p<0.05 vs UC) while procaine administration significantly increased the expression of this inhibitor (p<0.05 vs OB). Ribosomal 18S expression was used as housekeeping.</p

Non cytotoxic effect of Procaine.

<p>The effect of several doses of procaine (0.5, 1 and 2 μM) on cell proliferation and viability was studied onundifferentiated mesenchymal stem cells (UC) (A), MSC plus Procaine 0.5 μM (B), 1 μM (C) y 2 μM (D) for 48h, 7 and 21 days of treatment. Changes in cell proliferation and viability were also studied on MSC differentiation into osteo/odontoblasts (E) at 48h, 14d and 21d. The effects of procaine on cell proliferation and viability on these osteo/odontoblasts were studied at 0.5 μM (F), 1 μM (G) and 2 μM (H). * p<0.001 vs 48h and # p<0.001 vs 7d.</p

Procaine decreases mineralization of osteo/odontoblasts differentiated from mesenchymal stem cells (MSC).

<p>A) Calcium content and B) alkaline phosphatase activity were significantly increased after osteo/odontogenic stimulus (OB) (* p< 0.05 vs. undifferentiated cells (UC)) at 21 days. Procaine addition (OB+Proc) significantly decreased calcium content and alkaline phosphatase activity after 21 days of differentiation (• p< 0.05 vs. OB cells). C) Alizarin red staining increased after osteo/odontoblasts differentiation (OB) while procaine addition (OB+Proc) significantly decreased mineralization during the differentiation process. Images are representative of three cultures. D) MGP gene expression was increased after procaine administration, impairing the mineralization process in OB cells. The size and intensity of amplicon was analyzed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping.</p

Procaine also promotes changes on mature osteo/odontoblasts.

<p>Procaine addition for 10 days to differentiated osteoblasts cells from MSC for 31days led to a decrease in A) alkaline phosphatase activity (*** p<0.001, ** p<0.01 vs. undifferentiated cells (UC) and ## p<0.01 vs OB cells) and B) on osteo/odontogenic genes (***p<0.001 vs UC cells and ### p<0.001 vs. OB cells). C) Calcium content and D) alizarin red staining did not change with respect to OB cells after 31 days of osteo/odontogenic stimulus and the last 10 days with procaine. Images representative of three experiments.</p