Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity

Abstract

Osteocytes are the most abundant osteoblast lineage cells within the bone matrix. They respond to mechanical stimulation and can participate in the release of regulatory proteins that can modulate the activity of other bone cells. We hypothesize that neuropeptide Y (NPY), a neurotransmitter with regulatory functions in bone formation, is produced by osteocytes and can affect osteoblast activity. To study the expression of NPY by the osteoblast lineage cells, we utilized transgenic mouse models in which we can identify and isolate populations of osteoblasts and osteocytes. The Col2.3GFP transgene is active in osteoblasts and osteocytes, while the DMP1 promoter drives green fluorescent protein (GFP) expression in osteocytes. Real-time PCR analysis of RNA from the isolated populations of cells derived from neonatal calvaria showed higher NPY mRNA in the preosteocytes/ osteocytes fraction compared to osteoblasts. NPY immunostaining confirmed the strong expression of NPY in osteocytes (DMP1GFP + ), and lower levels in osteoblasts. In addition, the presence of NPY receptor Y1 mRNA was detected in cavaria and long bone, as well as in primary calvarial osteoblast cultures, whereas Y2 mRNA was restricted to the brain. Furthermore, NPY expression was reduced by 30-40% in primary calvarial cultures when subjected to fluid shear stress. In addition, treatment of mouse calvarial osteoblasts with exogenous NPY showed a reduction in the levels of intracellular cAMP and markers of osteoblast differentiation (osteocalcin, BSP, and DMP1). These results highlight the potential regulation of osteoblast lineage differentiation by local NPY signaling. KeywordsNeuropeptide Y; Osteocytes; Osteoblasts; GFP; Bone Neuropeptide Y (NPY) is a 36-amino acid polypeptide that belongs to the larger family of neuropeptides, which also includes the pancreatic polypeptide and the peptide YY Allen et al., 1992]. NPY signals through a class of receptors known as Y receptors, members of the G-protein-coupled receptors [Lemos et al., 1997;Raimondi et al., 2002]. The Y receptor system consists of five Y receptors; Y1, Y2, Y4, Y5, and Y6 (which is present only in the mouse). NPY is highly expressed in the hypothalamus, and its receptors Y1, Y2, and Y5 are found in the central nervous system, including the hypothalamus [Sar et al., 1990;Parker and Herzog, 1999].Functionally, NPY is a potent orexigenic peptide; its expression is up-regulated in the hypothalamus of experimental diabetic rats la Fleur et al., 2003]. NPY acts through the Y2 receptor in the hypothalamus to centrally regulate bone mass [Baldock et al., 2002]. This observation was reinforced using germline deletion of Y2 or Y1 receptors in mice, resulting in a higher trabecular bone volume and elevated cortical bone mass [Baldock et al., 2005]. Moreover, targeted deletion of Y2 in the hypothalamus revealed an increased bone volume comparable to the effect of germ line deletion of Y1 or Y2 in mice, thus confirming the Y2-mediated central regulation of bone mass [Baldock et al., 2005]. In...

Critical role for Y1 receptors in mesenchymal progenitor cell differentiation and osteoblast activity

“…The lineage and maturation-stage specificity of the 2.3ColCre;Y1 lox/lox model alleviates such potential confounders and produces a clearer indication of the role of Y1 in mature osteoblastic cells. As such, the repressive action of NPY on osteoblast function described by the 2.3ColCre;Y1 lox/lox studies is consistent with known actions of NPY on osteoblasts in vitro (4,7) and in vivo. (4,39) Cultures generated from single-cell suspensions of marrow have shown that BMSCs are clonogenic and grow from foci before becoming confluent.…”

“…Y1 expression has been shown to change with maturation of osteoblastic cells, increasing with expression of late-stage markers such as dentin matrix protein 1 (DMP1). (7) Indeed, mechanical loading, a process restricted to terminally differentiated osteoblastic cells, reduced NPY expression, consistent with Y1 signaling occurring in a maturation-specific manner. Additionally, it is also possible that the decreased total mineralization observed in BMSC cultures from germ line Y1 À/À mice may relate to indirect effects caused by the global deletion of the Y1 receptor, such as deficiency of Y1 receptors from other cells types in the heterogeneous BMSC cultures.…”

“…(10) In addition, administration of NPY to BMSC and calvarial cultures isolated from wild-type mice markedly reduced cell numbers (4) and markers of osteoblast differentiation. (7) respectively. Interestingly, the inhibitory effect of NPY on cell number was absent in cells isolated from germ-line Y1 receptor knockout (Y1 À/À ) mice, (4) indicating a direct effect of NPY on bone cells via the Y1 receptor.…”

“…(4,5) However, the mechanism behind the action of the Y1 versus the Y2 receptor on bone differs: Conditional deletion of hypothalamic Y2 receptors recapitulates the phenotype of germ-line Y2 receptor knockout ORIGINAL ARTICLE J JBMR (Y2 À/À ) mice, (5) whereas bone homeostasis is unaltered by hypothalamus-specific deletion of the Y1 receptor. (4) Y1 receptor expression has been demonstrated in osteoblastic cells lining endocortical and trabecular bone surfaces by in situ hybridization on femur sections and also by RT-PCR on RNA isolated from bone marrow stromal cell (BMSC) cultures (6) and primary calvarial cultures, (7) suggesting that the Y1 receptor may mediate effects on bone via direct actions on osteoblasts, whereas Y2 receptors could not be detected on bone cells using these methods.…”

Regulation of neuropeptide Y Y1 receptor expression by bone morphogenetic protein 2 in C2C12 myoblasts

“…However, the mechanism underlying the action of the Y1 receptor on bone differs from that of the Y2 receptor: both germline and conditional hypothalamic Y2 receptor knockout mice share the same high bone mass phenotype [9,10] demonstrating that central hypothalamic Y2 receptors are crucial for this process, whereas bone tissue is unaltered by conditional deletion of hypothalamic Y1 receptors [4] indicating a nonhypothalamic control of bone mass. Y1 receptor expression has been demonstrated in osteoblastic cells lining endocortical and trabecular bone surfaces [11] and in primary calvarial cultures [12]. A recent study from osteoblast-specific Y1 receptor knockout mice revealed a high bone mass phenotype similar to the bone phenotype of germline Y1 receptor knockout mice, suggesting that the Y1 receptor mediates effects on bone via direct actions on osteoblasts and that peripheral Y1 receptors play a role in the regulation of bone formation [13].…”