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Pilose antler peptide promotes osteoblast proliferation, differentiation and mineralization via the insulin signaling pathway.

ABSTRACT: Osteoporosis is a severe bone disease characterized by a decrease in the density and structure of bones, with high risks of fractures. Pilose antler peptide (PAP), extracted and purified from deer antlers, can promote regeneration and fracture healing, and strengthen sinews and bone. To determine whether PAP can promote osteoblast development and to elucidate the molecular mechanisms underlying its functions, the present study investigated the effects of PAP on osteoblast proliferation, differentiation and mineralization, and the role of the insulin signaling pathway using MTT assay, alkaline phosphatase activity assay, western blot analysis and reverse transcription-quantitative PCR. The present results suggested that PAP promoted osteoblast proliferation, differentiation and mineralization in vitro via the insulin signaling pathway. The effect of PAP on insulin signaling in osteoblasts may be mediated via the ERK pathway and partially by the PI3K/Akt pathway. The present results indicated that PAP could potentially be developed as an alternative treatment strategy for bone diseases related to diabetes characterized by insulin signaling impairment.

SUBMITTER: Yun C

PROVIDER: S-EPMC6966112 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Pilose antler peptide promotes osteoblast proliferation, differentiation and mineralization via the insulin signaling pathway.

Yun Changjun C Qian Wenjie W Wu Junyi J Yuan Chenxi C Jiang Songzhou S Lv Jinpeng J

Experimental and therapeutic medicine 20191205 2

Osteoporosis is a severe bone disease characterized by a decrease in the density and structure of bones, with high risks of fractures. Pilose antler peptide (PAP), extracted and purified from deer antlers, can promote regeneration and fracture healing, and strengthen sinews and bone. To determine whether PAP can promote osteoblast development and to elucidate the molecular mechanisms underlying its functions, the present study investigated the effects of PAP on osteoblast proliferation, differen ...[more]

PMID: 32010253

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Project description:Bone loss occurs as a consequence of a variety of diseases as well as from traumatic injuries, and often requires therapeutic intervention. Strategies for repairing and replacing damaged and/or lost bone tissue include the use of biomaterials and medical implant devices with and without osteoinductive coatings. The soluble growth factor angiopoietin-1 (Ang-1) has been found to promote cell adhesion and survival in a range of cell types including cardiac myocytes, endothelial cells and fibroblasts through an integrin-dependent mechanism. Furthermore, the short sequence QHREDGS has been identified as the integrin-binding sequence of Ang-1 and as a synthetic peptide has been found to possess similar integrin-dependent effects as Ang-1 in the aforementioned cell types. Integrins have been implicated in osteoblast differentiation and bone mineralization, processes critical to bone regeneration. By binding integrins on the osteoblast surface, QHREDGS could promote cell survival and adhesion, as well as conceivably osteoblast differentiation and bone mineralization. Here we immobilized QHREDGS onto polyacrylate (PA)-coated titanium (Ti) plates and polyethylene glycol (PEG) hydrogels. The osteoblast differentiation marker, alkaline phosphatase, peaked in activity 4-12 days earlier on the QHREDGS-immobilized PA-coated Ti plates than on the unimmobilized, DGQESHR (scrambled)- and RGDS-immobilized surfaces. Significantly more bone matrix was deposited on the QHREDGS-immobilized Ti surface than on the other surfaces as determined by atomic force microscopy. The QHREDGS-immobilized hydrogels also had a significantly higher mineral-to-matrix (M/M) ratio determined by Fourier transform infrared spectroscopy. Alizarin Red S and von Kossa staining and quantification, and environmental scanning electron microscopy showed that while both the QHREDGS- and RGDS-immobilized surfaces had extensive mineralization relative to the unimmobilized and DGQESHR-immobilized surfaces, the mineralization was more considerable on the QHREDGS-immobilized surface, both with and without the induction of osteoblast differentiation. Finally, treatment of cell monolayers with soluble QHREDGS was demonstrated to upregulate osteogenic gene expression. Taken together, these results demonstrate that the QHREDGS peptide is osteoinductive, inducing osteoblast differentiation, bone matrix deposition and mineralization.

Project description:Eucommia leaves are dry leaves of Eucommia ulmoides which have long been considered as a functional health food for the treatment of hypertension, hypercholesterolemia, fatty liver, and osteoporosis. With the recent development of Chinese medicine, Eucommia leaves are widely used for tonifying the kidneys and strengthening bone. However, the specific molecular mechanism of Eucommia leaves for strengthening bone remains largely unknown. Osteoblasts are the main functional cells of bone formation; thus, it is essential to study the effect of Eucommia leaves on osteoblasts to better understand their mechanism of action. In the present study, we prepared an aqueous extract of Eucommia leaves (ELAE) and determined its content by high-performance liquid chromatography (HPLC). The effects of ELAE on MC3T3-E1 cells were investigated by CCK-8 assay, alkaline phosphatase (ALP), and Alizarin red S staining assays, combined with RNA sequencing (RNA-seq) and qRT-PCR validation. We demonstrated that ELAE had a significant promoting effect on the proliferation of MC3T3-E1 cells and significantly enhanced extracellular matrix synthesis and mineralization, which were achieved by regulating various functional genes and related signaling pathways. ELAE significantly increased the expression level of genes promoting cell proliferation, such as Rpl10a, Adnp, Pex1, Inpp4a, Frat2, and Pcdhga1, and reduced the expression level of genes inhibiting cell proliferation, such as Npm1, Eif3e, Cbx3, Psmc6, Fgf7, Fxr1, Ddx3x, Mbnl1, and Cdc27. In addition, ELAE increased the expression level of gene markers in osteoblasts, such as Col5a2, Ubap2l, Dkk3, Foxm1, Col16a1, Col12a1, Usp7, Col4a6, Runx2, Sox4, and Bmp4. Taken together, our results suggest that ELAE could promote osteoblast proliferation, differentiation, and mineralization and prevent osteoblast apoptosis. These findings not only increase our understanding of ELAE on the regulation of bone development but also provide a possible strategy to further study the prevention and treatment of osteogenic related diseases by ELAE.

Project description:BackgroundDeer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration.MethodsTo identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies.ResultsCell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10.ConclusionUsing a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.

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Pilose antler peptide promotes osteoblast proliferation, differentiation and mineralization via the insulin signaling pathway.

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Pilose antler peptide promotes osteoblast proliferation, differentiation and mineralization via the insulin signaling pathway.

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