Korean J Physiol Pharmacol.  2021 May;25(3):197-206. 10.4196/kjpp.2021.25.3.197.

Carnosol induces the osteogenic differentiation of bone marrowderived mesenchymal stem cells via activating BMP-signaling pathway

• Abdallah BM

• KMID: 2514945
• DOI: http://doi.org/10.4196/kjpp.2021.25.3.197

Abstract

Carnosol is a phenolic diterpene phytochemical found in rosemary and sage with reported anti-microbial, anti-oxidant, anti-inflammatory, and anti-carcinogenic activities. This study aimed to investigate the effect of carnosol on the lineage commitment of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblasts and adipocytes. Interestingly, carnosol stimulated the early commitment of mBMSCs into osteoblasts in dose-dependent manner as demonstrated by increased levels of alkaline phosphatase activity and Alizarin red staining for matrix mineralization. On the other hand, carnosol significantly suppressed adipogenesis of mBMSCs and downregulated both early and late markers of adipogenesis. Carnosol showed to induce osteogenesis in a mechanism mediated by activating BMP signaling pathway and subsequently upregulating the expression of BMPs downstream osteogenic target genes. In this context, treatment of mBMSCs with LDN-193189, BMPR1 selective inhibitor showed to abolish the stimulatory effect of carnosol on BMP2-induced osteogenesis. In conclusion, our data identified carnosol as a novel osteoanabolic phytochemical that can promote the differentiation of mBMSCs into osteoblasts versus adipocytes by activating BMP-signaling.

Keyword

Adipocyte; Bone morphogenetic protein signaling; Carnosol; Mesenchymal stem cells; Osteoblast

Figure

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  Fig. 1 Carnosol induces the differentiation of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) into osteoblasts. (A) Studying the cytotoxicity of carnosol on primary culture of mBMSCs using MTT cell viability assay. (B) Studying the effect of different concentrations of carnosol on mBMSCs cell proliferation as measured by counting cell number. Cells were either non-treated (0) or treated with different doses of carnosol for 3 days. (C) Stimulatory effect of carnosol on osteoblast differentiation of mBMSCs as assessed by ALP activity quantification and (D) Alizarin red staining quantification for matrix mineralization after 6 and 12 days of induction respectively. Stained images were shown under each graph. mBMSCs were either non-induced (control, ctrl), or induced with OIM in the absence (I) or the presence of different concentrations of carnosol. Values were shown as fold change over control. Values are mean ± standard deviation of three independent experiments (*p < 0.05, **p < 0.005 compared to Control [0] for [A and B] and compared to differentiated cells without carnosol [I] for panals [C and D]). ALP, alkaline phosphatase; OIM, Osteogenic-induction medium.

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  Fig. 2 Carnosol induces the expression of osteoblast-related genes. (A) Carnosol upregulated the gene expression of early (Runx2, Osx, Alp, and Col1a1) and late osteogenic factors (Ocn and Opn) in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) during osteogenesis. mBMSCs were induced to osteoblast differentiation using OIM in the absence (I+DMSO) or the presence of 10 µM carnosol (I+Carnosol) for 12 days. Gene expression values were normalization to reference genes and represented as fold change over induced cells without carnosol. (B) Analysis of carnosol-induced osteoblast-related genes in mBMSCs during osteogenesis using qPCR-based osteogenic gene array assay. The pie chart showed the percentage of upregulated genes as categorized by their osteogenic functions according to the data in Table 1. Values are mean ± standard deviation of three independent experiments (**p < 0.005 compared to I+DMSO). OIM, Osteogenic-induction medium.

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  Fig. 3 Carnosol inhibits the differentiation of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) into adipocytes. (A) Inhibitory effect of carnosol on adipogenesis of mBMSCs as measured by Oil Red O staining for fat droplets formation. mBMSCs were induced to differentiated into adipocytes either in the presence of DMSO (I) as control or different concentrations of carnosol. Scale bars = 100 µm. (B) Quantification of Oil Red O staining after 12 days of differentiation. (C) Quantitative real-time PCR analysis of the adipogenic specific genes expression in the differentiated mBMSCs into adipocytes in the presence of different concentrations of carnosol. Gene expression values were normalized to reference genes and represented as fold inhibition to induced cells without carnosol. Values are mean ± standard deviation of three independent experiments (*p < 0.05, **p < 0.005, compared to [I]).

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  Fig. 4 Carnosol promotes bone morphogenetic protein (BMP)-induced osteogenesis in mouse bone marrow-derived mesenchymal stem cells (mBMSCs). (A) Studying the effect of carnosol on promoting the osteogenic induction of different osteogenic growth factors in mBMSCs. Cells were induced for osteogenesis without (I+DMSO) or with TGFβ1 (10 ng/ml), BMP2 (100 ng/ml), IGF1 (100 ng/ml), bFGF (100 ng/ml) or PDGF (100 ng/ml) for 6 days in the absence or the presence of 10 µM carnosol for 6 days. (B) Effect of carnosol on BMP2, 4, and 7-induced osteogenesis in mBMSCs as measured by quantitative Alizarin Red staining for matrix mineralization after 12 days of induction. Representative images of Alizarin Red staining were shown. (C) Effect of carnosol on inducing the expression of BMP2-upregulated osteogenic gene expression. Gene expression values were normalization to reference genes and represented as fold change over induced cells without carnosol. Values are mean ± standard deviation of three independent experiments (**p < 0.005, compared to induced cells without carnosol [I+DMSO]).

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  Fig. 5 Carnosol promotes osteoblast differentiation of mouse bone marrow-derived mesenchymal stem cells (mBMSCs) through the activation of bone morphogenetic protein (BMP) signaling pathway. (A) Western blot analysis showing the dose-dependent stimulatory effect of carnosol (10 µM) on the activation of Smad1/5/8 phosphorylation in mBMSCs after 10 min. (B) Western blot analysis demonstrating the ability of BMP1R inhibitor (LDN-193189, 10 µM) to inhibit the stimulatory effect of carnosol on BMP-induced Smad1/5/8 phosphorylation in mBMSCs after 10 min of treatment. (C) BMP1R inhibitor (LDN-193189, 10 µM) suppressed the stimulatory effect of carnosol (10 µM) on BMP2-induced osteogenesis in mBMSCs as measured by quantitative ALP activity. Cells were pre-treated with LDN-193189, 10 µM and induced with BMP2 in the absence or the presence of carnosol (10 µM) for 6 days. (D) Schematic diagram illustrating the mechanism of the stimulatory effect of carnosol to on osteogenesis via activating canonical BMP signalling. Values are mean ± standard deviation of three independent experiments (**p < 0.005). ALP, alkaline phosphatase.

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