Increased extracellular and intracellular Ca²⁺ lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms

Highlights

• Both high [Ca²⁺]_o and high [Ca²⁺]_i enhanced adipocyte accumulation in BMSCs.

• High [Ca²⁺]_i enhanced the proliferation of BMSCs but not adipocyte differentiation.

• High [Ca²⁺]_o suppressed the phosphorylation of ERK in BMSCs.

• Inhibition of ERK enhanced the differentiation of BMSCs into adipocytes.

• High [Ca²⁺]_o-mediated suppression of ERK may be a new therapy target for anemia.

Abstract

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca²⁺ ([Ca²⁺]_o and [Ca²⁺]_i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca²⁺]_o and high [Ca²⁺]_i enhanced adipocyte accumulation, which suggested that increases in [Ca²⁺]_o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca²⁺]_o and high [Ca²⁺]_i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca²⁺]_i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca²⁺]_o (addition of CaCl₂) leads to increases in [Ca²⁺]_i. Flow cytometric methods revealed that high [Ca²⁺]_o suppressed the phosphorylation of ERK independently of intracellular Ca²⁺. The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca²⁺ provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca²⁺, which results in BMSC proliferation.

Introduction

Bone marrow-derived mesenchymal stem cells that are found among bone marrow stromal cells (BMSCs) are the common progenitors for both adipocytes and osteoblasts [1], [2], [3], [4]. Clinically, increases in marrow adipogenesis are known to be associated with osteoporosis, diabetes mellitus, and age-related osteopenia [5]. We recently reported that upon treatment of BMSCs with insulin and dexamethasone, high extracellular Ca²⁺ ([Ca²⁺]_o) enhanced adipocyte but not osteoblast accumulation in BMSCs. These observations suggested that the increases in [Ca²⁺]_o that are caused by bone resorption may accelerate adipocyte accumulation rather than osteoblastic bone formation in aging and/or diabetic patients [6].

Both extracellular and intracellular Ca²⁺ are versatile signaling molecules that are involved in the regulation of a number of cell functions, including proliferation, differentiation, and cell death. In the previous study, we showed that both high [Ca²⁺]_o and intracellular Ca²⁺ ([Ca²⁺]_i) enhance the accumulation of adipocytes. However, the molecular mechanisms by which high [Ca²⁺]_o and high [Ca²⁺]_i may mediate an increase in the adipogenic induction of lipid accumulation remain unclear. In the process of accumulation of bone marrow adipocytes, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. In this study, we show that enhanced adipocyte accumulation by high [Ca²⁺]_i is caused by enhanced proliferation of BMSCs but not enhanced differentiation. We also show that high [Ca²⁺]_o enhances both the proliferation of BMSCs through increment of [Ca²⁺]_i and the adipocyte differentiation independently of increased [Ca²⁺]_i, which result in adipocyte accumulation. Although the involvement of mitogen-activated protein kinases (MAPKs) in the regulation of osteogenic differentiation has been demonstrated in osteoblasts and mesenchymal stem cells [7], [8], [9], the role of MAPK pathways in the regulation of bone marrow adipogenic differentiation by Ca²⁺ remains unclear. Here, we show that increased extracellular Ca²⁺ but not intracellular Ca²⁺ enhances BMSC adipocyte differentiation through the suppression of extracellular-signal-regulated kinase (ERK) activity. These findings regarding enhanced adipogenic differentiation and subsequent inhibition of osteogenic differentiation by high extracellular Ca²⁺ via suppression of the ERK pathway may have the potential to be the target for treatment of osteopenia and anemia.