Mechanistic Roles of Leptin in Osteogenic Stimulation in Thoracic Ligament Flavum Cells

Abstract

Obesity is a risk factor for thoracic ossification of ligament flavum (TOLF) that is characterized by ectopic bone formation in the spinal ligaments. Hyperleptinemia is a common feature of obese people, and leptin, an adipocyte-derived cytokine with proliferative and osteogenic effects in several cell types, is believed to be an important factor in the pathogenesis of TOLF. However, how leptin might stimulate cell osteogenic differentiation in TOLF is not totally understood. We reported here that leptin-induced osteogenic effect in TOLF cells is associated with activation of signaling molecules STAT3, JNK, and ERK1/2 but not p38. Blocking STAT3 phosphorylation with a selective inhibitor, AG490, significantly abolished leptin-induced osteogenic differentiation of TOLF cells, whereas blocking ERK1/2 and JNK phosphorylation with their selective inhibitors PD98059 and SP600125, respectively, had only marginal effects. In addition, we showed that STAT3 interacted with Runt-related transcription factor 2 (Runx2) in the nucleus, and STAT3, Runx2, and steroid receptor coactivator steroid receptor coactivator-1 were components of the transcription complex recruited on Runx2 target gene promoters in response to leptin treatment. Our experiments identified STAT3, Runx2, and steroid receptor coactivator-1 as critical molecules in mediating leptin-stimulated cell osteogenesis in TOLF. Ossification of ligament flavum (OLF)3 of the spine is characterized by a heterotopic bone formation in the ligament flavum that is normally composed of fibrous tissues (1). Ossification could enlarge the spinal canal and compresses the spinal cord, resulting in serious neurological damages. Epidemiology has shown that high incidence rate of OLF occurs in thoracic spine (2). It has been documented that obesity represents a risk factor for thoracic ossification of ligament flavum (TOLF), particularly in Asian people (3). Indeed, hereditary obese rats, Zucker fatty (fa/fa) rats, are prone to OLF (4). A common feature of obese people is hyperleptinemia (5). Leptin, an adipocyte-derived cytokine, can stimulate the proliferation and osteogenic differentiation of various cell lines, such as the embryonic cell line C3H10T1/2, human NHOst cells, and human osteoblastic cells (6, 7). However, the molecular mechanism underlying the osteogenic effect of leptin in TOLF is not totally understood.Leptin exerts its biological activity through binding to its receptors, which belong to cytokine receptor superfamily. Different leptin receptor isoforms exist, including a long form (ObRb) and a short form (ObRa) (8). In vitro and in vivo studies have shown that leptin activates cytokine-like signal transduction via the long form receptor. Upon leptin stimulation, intracellular Janus tyrosine kinases are activated via transphosphorylation and phosphorylate tyrosine residues on the long form leptin receptor and on signal transducers and activators of transcription (STAT) proteins (9). Phosphorylated STAT proteins dimerize and translocate to the nucleus to activate ge...

Growth/differentiation Factor-5 Induces Osteogenic Differentiation of Human Ligamentum Flavum Cells through Activation of ERK1/2 and p38 MAPK

“…Recent studies have demonstrated that the activation of MAPK signaling cascade contributes to osteogenic differentiation of cultured LFCs induced by mechanical stress and leptin, which were considered as the causes of OLF [25,43]. However, whether the activation of ERK1/2 and p38 MAPK is involved in rhGDF-5 induced LFCs differentiation need to be further studied.…”

“…), 100 U/ml penicillin and 100 pg/ml streptomycin, and incubated in 95% air/5% CO 2 atmosphere at 37 °C, LFCs were harvested with 0.2% trypsin (Sigma-Aldrich)/0.02% ethylenediamine tetraacetic acid once they migrated from the explants and became confluent. The third passage LFCs were used in the following studies [25].…”

Association between serum leptin and bone metabolic markers, and the development of heterotopic ossification of the spinal ligament in female patients with ossification of the posterior longitudinal ligament

“…To date, a few in vitro studies have revealed the mechanisms by which leptin induces osteogenic differentiation in spinal ligament cells [6,24]. Fan et al [6] demonstrated that leptin caused significant increases in mRNA expression of alkaline phosphatase (ALP) and osteocalcin in thoracic ossification of ligament flavum (TOLF) cells, but not in non-TOLF cells, and that the effect was both dose-and time-dependent.…”

“…Fan et al [6] demonstrated that leptin caused significant increases in mRNA expression of alkaline phosphatase (ALP) and osteocalcin in thoracic ossification of ligament flavum (TOLF) cells, but not in non-TOLF cells, and that the effect was both dose-and time-dependent. These findings suggest that TOLF cells are considerably more sensitive to leptin stimulation and that leptin has the potential to promote osteogenic differentiation in ligament flavum cells.…”

Effect of CXCR4 inhibitor AMD3100 on alkaline phosphatase activity and mineralization in osteoblastic MC3T3-E1 cells

“…Similarly, our results show that blocking of SDF-1/CXCR4 signaling only had an effect on phosphorylation of STAT3 at the early stage of osteogenic differentiation in MC3T3-E1 cells. Furthermore, STAT3 and Runx2 are two of the major transcription factors that play essential roles in osteogenesis, they interact with each other in the nucleus, and Runx2 is a master regulator at the early phase of osteogenic differentiation; this interaction might affect osteogenic regulation (33)(34)(35). Further studies are needed to explain whether the interplay of STAT3 and Runx2 are involved in STAT3 signaling at the early stage of osteogenic differentiation.…”