Elsevier

Life Sciences

Volume 94, Issue 1, 14 January 2014, Pages 45-53
Life Sciences

Involvement of MAPK signaling pathway in the osteogenic gene expressions of Cervi Pantotrichum Cornu in MG-63 human osteoblast-like cells

https://doi.org/10.1016/j.lfs.2013.11.002Get rights and content

Abstract

Aims

The purposes of this study were to determine whether Cervi Pantotrichum Cornu (CPC) has osteogenic activities in human osteoblastic MG-63 cells and to investigate the underlying molecular mechanism.

Main methods

The effects of CPC on alkaline phosphatase activity, collagen synthesis, and calcium deposits were measured. The COL1A1, ALPL, BGLAP, and SPP1 expressions were measured by real-time PCR. Phosphorylated MAP kinases (ERK1/2, JNK1/2, p38, ELK1, and cJUN) were studied by western blot analysis. The involvement of MAPK pathway in osteogenic gene expressions was determined by using each selective MAPK inhibitor (PD98059, SP600125, and SB203580).

Key findings

CPC increased alkaline phosphatase activity, collagen synthesis, and calcium deposits. CPC activated ERK1/2, JNK1/2, p38, and ELK1 phosphorylation except cJUN. CPC increased the COL1A1, ALPL, BGLAP, and SPP1 gene expressions. The elevated COL1A1 and BGLAP expressions were inhibited by PD98059, SP600125 or SB203580. The elevated ALPL expression was blocked by SB203580. The elevated SPP1 expression was inhibited by SP600125 or SB203580. CPC increased COL1A1 and BGLAP expressions via ERK1/2, JNK1/2, and p38 MAPKs pathways and SPP1 expression via JNK1/2 and p38 pathways. p38 pathway is needed for ALPL expression.

Significance

These results imply that MAPK signaling pathway is an indispensable factor for bone matrix genes expression of CPC in MG-63 human osteoblast-like cells.

Introduction

Cervi Pantotrichum Cornu (CPC) is the young pilose antler of a male Cervus nippon Temminck or Cervus elaphus Linnaeus, and is renowned as a bone-strengthening drug in many Asian countries. It is widely used in traditional medicinal practices to promote virility, replenish vital essence and blood, and strengthen the tendons and bones (Bensky et al., 2004).

The osteoblasts serve the function of synthesizing the extracellular matrix (ECM) of bone, regulating calcium deposition and mineralization, and responding to mechanical stimuli. Accordingly, osteoblasts are believed to play a pivotal role in strengthening bone (Salgado et al., 2004).

Recent studies have also demonstrated that mechanotransduction in bone cells involves the sequential activations (via phosphorylation cascade) of various intracellular signaling molecules, including mitogen-activated protein kinases (MAPKs) (Greenblatt et al., 2013, He et al., 2012, Mahalingam et al., 2013, Thouverey and Caverzasio, 2012), phosphoinositide 3-kinase (PI3k)/Akt (Danciu et al., 2003), and protein kinases B and C (Biggs et al., 1999, Geng et al., 2001). As a result, mechanical signals can activate transcription factors such as activator protein-1 (AP-1) (Peverali et al., 2001), bone-specific transcriptional regulator (Cbfa1) (Franceschi, 1999, Wu et al., 2012), and NF-kB (Granet et al., 2001) to modulate the expression of genes that regulate different physiological functions (Wu et al., 2006).

There are several studies about the effects of CPC. Ahn and Shim studied the effects of CPC on an aged ovariectomized rat model of postmenopausal osteoporosis in 1998 (Ahn and Shim, 1998) and Lee et al. showed the stimulating effects of fermented CPC on osteoblastic differentiation and mineralization (Lee et al., 2011). However, merely identifying the drug's fragmentary effects is not sufficient. The evaluation of the action mechanisms of a medicine is also important to verify the efficacy of the drug.

In the present study, we tried to identify the MAPKs signaling pathway among numerous mechanisms (extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38). The expression of osteogenic genes including collagen, type I, alpha 1 (COL1A1), alkaline phosphatase (ALPL), bone gamma-carboxyglutamic acid protein (BGLAP, osteocalcin), and secreted phosphoprotein 1 (SPP1, osteopontin) were measured by real-time PCR. The signaling pathways of osteogenic mRNAs expressions were verified by using PD98059 (selective inhibitor of ERK1/2 and ELK1), SP600125 and BI-78D3 (selective inhibitors of JNK1/2), and SB203580 (selective inhibitor of p38).

Section snippets

Materials

The CPC was obtained from Hmax Co. (Jecheon, Chungbuk, Korea) and it had been authenticated by Prof. Bu (the department of herbal pharmacology, Kyung-Hee University). 100 g of CPC was extracted in 2,000 ml of distilled water (DW) at 100 °C for 3 h, filtered through filter paper, and concentrated by rotary evaporator and then freeze-dried. The yield of freeze-dried CPC was calculated to be 29.9%. Anti-JNK, anti-p-JNK, anti-ERK, anti-p-ERK, anti-cJUN, anti-p-cJUN, anti-p-ELK-1, anti-beta actin

Cell proliferation

The effects of CPC on the cell proliferation of MG-63 cells were examined. The cell proliferations of MG-63 cells treated with PBS showed 100.0 ± 3.8% and CPC (5, 10, 25, and 50 μg/ml)-treated cells showed 99.1 ± 6.8%, 95.5 ± 3.8%, 108.7 ± 0.9%, and 106.3 ± 3.2% of cell proliferation. Cell proliferations of 25 and 50 μg/ml of CPC-treated groups were significantly increased (P < 0.05, Fig. 1A).

Alkaline phosphatase activity

The ALP activity of MG-63 cells treated with PBS showed 100.0 ± 0.3% and the cells treated with CPC (5, 10, 25, and 50 

Discussion

There is considerable scientific evidence for the osteogenic effects of CPC. Zhou showed that the polypeptides from CPC promoted proliferation of chondrocytes and osteoblast precursors and fracture healing (Zhou et al., 1999). The enzymatic hydrolysis of CPC enhanced the proliferative activity toward UMR-106 osteoblast cells (Zheng et al., 2010). Shi studied the effect of CPC on corticosteroid-induced avascular necrosis of the femoral head in rats (Shi et al., 2010). The effects on osteoporosis

Conclusion

The present study has shown that CPC-treatment showed the osteogenic activities and increased bone matrix genes via MAPK signaling pathway. COL1A1 and BGLAP gene expressions need all of MAPKs (ERK1/2, JNK1/2 and p38) phosphorylation and SPP1 gene expression needs both JNK1/2 and p38 phosphorylation (Fig. 7). ALPL gene expression needs p38 phosphorylation (Fig. 7). These results could provide a mechanistic explanation for the bone-strengthening effects of CPC.

The following are the supplementary

Conflict of interest statement

The authors report no conflict of interest.

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