The calcium transient characteristics induced by fluid shear stress affect the osteoblast proliferation

https://doi.org/10.1016/j.yexcr.2017.11.001Get rights and content

Highlights

  • External Ca2+ determines FSS-induced osteoblast proliferation and differentiation.

  • The mechanism relies on the mitochondrial energetic metabolism.

  • MCU enhanced TCA cycle and subsequently affecting cell cycle proteins.

  • FSS-induced intracellular calcium transients was crucial to the cell proliferation.

Abstract

Ca2+ signaling is essential for bone metabolism. Fluid shear stress (FSS), which can induce a rapid release of calcium from endoplasmic reticulum (ER) to produce calcium transients, plays a significant role in osteoblast proliferation and differentiation. However, it is still unclear of how calcium transients induced by FSS activating a number of downstream signals which subsequently regulate cell functions. In this study, we performed a group of Ca2+ transients models, which were induced by FSS to investigate the effects of different magnitudes of Ca2+ transients in osteoblast proliferation. Further, we performed a global proteomic profile of MC3T3-E1 cells in different Ca2+ transients models stimulated by FSS. GO enrichment and KEGG pathway analysis revealed that the TCA cycle was activated in the proliferating process. The activation of TCA needed mitochondrial Ca2+ uptake which were influenced by the amplitude of Ca2+ transients induced by FSS. Our work elucidate that osteoblast proliferation induced by FSS was related to the magnitude of calcium transients, which further activated energetic metabolism signaling pathway. This work revealed further understanding the mechanism of osteoblast proliferation induced by mechanic loading and help us to design new methods for osteoporosis therapy.

Introduction

Fluid shear stress (FSS) affected both proliferation and differentiation of osteoblasts [1], [2], [3] However, the detailed molecular mechanism is not completely clarified. FSS can mechanically stimulate cells, and this stimulus is then translated into biochemical signals that exert biological effects in cells [4]. Ca2+ signaling is the first rapid response to FSS [5]. Upon FSS stimulation, intracellular calcium transients arise in osteoblasts, which has been reported to be essential for cell biological functions including differentiation, gene expression and cell proliferation [6]. The amplitude, frequency and duration of calcium transients played significant roles in regulating cell functions [7], [8], [9], [10]. Despite of intensive studies, how calcium transients activate the downstream signals which subsequently regulate cell functions is still unclear.

Energetic metabolism plays also an important role in cell proliferation and differentiation. Many of the biosynthetic activities of proliferating cells are involved in mitochondrial metabolism [11]. For example, the TCA cycle generates precursors to synthesize proteins, nucleic acids, and lipids, as well as ATP which provides energy for most of the cell biological process [12], [13]. It is obviously that the regulation of mitochondrial metabolism plays an important role in cell growth. Basso and Heersche [14] reviewed various mechanical stimuli and found that FSS was a major factor for affecting bone cell metabolism. Ca2+ uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca2+ levels, this flux influences energy production and could potentially alter intracellular [Ca2+]i signals [15]. However, calcium signaling, considered as the rapid response of FSS, is rarely reported on the effects of energetic metabolism inducing cell proliferation and differentiation.

Our previous study has demonstrated FSS induced MC3T3-E1 cells differentiation through ERK1/2 pathway in G0/G1 phase [16]. In the present study, we further explored the situation of osteoblast proliferation and differentiation induced by FSS aiming to understand the notable signaling pathways under different [Ca2+]i transient characteristics in the regulation of osteoblastic functions. We speculated that the characteristics of calcium transients induced by FSS might be the key regulator for osteoblast proliferation and differentiation, and the different models of calcium transients might induce diverged energy metabolism pathways to impact cell proliferation or differentiation. This helps us to further understand the mechanism of osteoblasts proliferation or differentiation induced by FSS, which is beneficial to device strategy for osteoporosis therapy.

Section snippets

Cell culture and G0/G1 phase arrest

The osteoblastic cell line MC3T3 -E1 was used for the majority of our experiments. MC3T3-E1 cells were cultured in α-MEM with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 g/ml streptomycin. Cells were maintained in a humidified incubator at 37 °C with 5% CO2/95% air and sub-cultured every 48 h. For FSS studies, 2 ml of 2.5 × 104/ml of cell suspension were seeded onto 22 × 76 mm poly-lysine coated glass slides. Cell cycle arrest in G0/G1 was achieved by serum deprivation. Cells were

Fluid shear stress induced MC3T3-E1 cells proliferation in calcium environment

MC3T3-E1 cells were synchronized by serum deprived for 24 h and then FSS (12 dyn/cm2) were loaded for 1 h in the existence of Ca2+ ranging from 1.8 to 7.2 mM. After stimulation, cells were cultured in normal media for another 24 h. Cell proliferation were determined by BrDU and ALP activity were assessed using ALP reagent kit. As shown in Fig. 1A, FSS in control (1.8 mM) and 2.4 mM increased ALP activity but did not increase cell numbers. Higher than 3.6 mM Ca2+ in the fluid flow increased cells

Discussion

Calcium (Ca2+) as a second messenger plays a vital role in cell functions. However, contradictory results have been reported on the functions of calcium transients on regulation of cell proliferation and differentiation [18], [19], [20], [21]. Fluid shear stress, which induced a rapid increase of calcium transients, is considered to be a potent mechanics factor to influence osteoblast proliferation and differentiation. However, the mechanism is not well explicated. In this study, we performed a

Conclusion

From the results above, we concluded that with the increase of [Ca2+]0, FSS induced different amplitudes of calcium transients, resulting in changes in energy metabolism pathways and thus affecting the cell proliferation and differentiation in different directions. FSS with > 3.6 mM [Ca2+]0 generated high amplitude transients which activated mitochondrial Ca2+ uptake. This process initiated the TCA cycle, which in turn activated down stream pathways involved in cell proliferation. Meanwhile, MCU

Acknowledgements

We are very grateful to the stuff of PTM-Biolabs (HangZhou) Co., Ltd. for their proteomics technical assistance, especially to Mr Fang zhang, and Dr. Xilong Wu. This work was supported by the National Natural Science Foundation of China (Project No. 31670954).

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