Calcitonin Receptor-positive Cells Research Articles

Gene expression profiling of peri-implant healing of PLGA-Li+ implants suggests an activated Wnt signaling pathway in vivo.

Bone development and regeneration is associated with the Wnt signaling pathway that, according to literature, can be modulated by lithium ions (Li+). The aim of this study was to evaluate the gene expression profile during peri-implant healing of poly(lactic-co-glycolic acid) (PLGA) implants with incorporated Li+, while PLGA without Li+ was used as control, and a special attention was then paid to the Wnt signaling pathway. The implants were inserted in rat tibia for 7 or 28 days and the gene expression profile was investigated using a genome-wide microarray analysis. The results were verified by qPCR and immunohistochemistry. Histomorphometry was used to evaluate the possible effect of Li+ on bone regeneration. The microarray analysis revealed a large number of significantly differentially regulated genes over time within the two implant groups. The Wnt signaling pathway was significantly affected by Li+, with approximately 34% of all Wnt-related markers regulated over time, compared to 22% for non-Li+ containing (control; Ctrl) implants. Functional cluster analysis indicated skeletal system morphogenesis, cartilage development and condensation as related to Li+. The downstream Wnt target gene, FOSL1, and the extracellular protein-encoding gene, ASPN, were significantly upregulated by Li+ compared with Ctrl. The presence of β-catenin, FOSL1 and ASPN positive cells was confirmed around implants of both groups. Interestingly, a significantly reduced bone area was observed over time around both implant groups. The presence of periostin and calcitonin receptor-positive cells was observed at both time points. This study is to the best of the authors' knowledge the first report evaluating the effect of a local release of Li+ from PLGA at the fracture site. The present study shows that during the current time frame and with the present dose of Li+ in PLGA implants, Li+ is not an enhancer of early bone growth, although it affects the Wnt signaling pathway.

MAP kinase pathways and calcitonin influence CD44 alternate isoform expression in prostate cancer cells

BackgroundDysregulated expression and splicing of cell adhesion marker CD44 is found in many types of cancer. In prostate cancer (PC) specifically, the standard isoform (CD44s) has been found to be downregulated compared with benign tissue whereas predominant variant isoform CD44v7-10 is upregulated. Mitogen-activated protein kinase pathways and paracrine calcitonin are two common factors linked to dysregulated expression and splicing of CD44 in cancer. Calcitonin has been found to increase proliferation and invasion in PC acting through the protein kinase A pathway.MethodsIn androgen-independent PC with known high CD44v7-10 expression, CD44 total and CD44v7-10 RNA or protein were assessed in response to exogenous and endogenous calcitonin and to inhibitors of protein kinase A, MEK, JNK, or p38 kinase. Benign cells and calcitonin receptor-negative PC cells were also tested.ResultsMEK or p38 but not JNK reduced CD44 total RNA by 40%–65% in cancer and benign cells. Inhibition of protein kinase A reduced CD44 total and v7-10 protein expression. In calcitonin receptor-positive cells only, calcitonin increased CD44 variant RNA and protein by 3 h and persisting to 48 h, apparently dependent on an uninhibited p38 pathway. Cells with constitutive CT expression showed an increase in CD44v7-10 mRNA but a decrease in CD44 total RNA.ConclusionThe MEK pathway increases CD44 RNA, while calcitonin, acting through the protein kinase A and p38 pathway, facilitates variant splicing. These findings could be used in the formulation of therapeutic methods for PC targeting CD44 alternate splicing.

Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: synergistic activation of differentiation, cell spreading, and fusion.

It was recently found that osteoblastic cells express TRANCE (tumor necrosis factor-related activation-induced cytokine), a newly identified member of the tumor necrosis factor superfamily, and that expression was increased by calciotropic hormones. Furthermore, soluble recombinant TRANCE induces osteoclast formation and resorption in stroma-free populations of hemopoietic precursor cells. However, overexpression of the decoy receptor osteoprotegerin in vivo shows that there are substantial differences in the sensitivity of different sites to resorption-inhibition, suggesting that either alternative ligands exist or the sensitivity of osteoclasts to TRANCE can be modified by cofactors. We therefore tested the possibility that cofactors might enhance osteoclast formation by TRANCE. We found that the number of tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive cells was increased by a factor of 10 by the presence of PGE2 in the absence of stromal cells. Moreover, although the tartrate-resistant acid phosphatase-positive cells that formed in TRANCE alone were typically mononuclear and poorly spread, the addition of PGE2 induced the formation of large, well spread multinuclear cells. There was an increase in bone resorption that corresponded with the increase in osteoclast number. PGE2 did not synergize with TRANCE for resorption-stimulation in mature cells. 8-Bromo-cAMP showed a similar syngergistic effect on osteoclastic differentiation. Thus, PGE2 appears to stimulate bone resorption through a direct effect on hemopoietic precursors, primarily through a synergistic effect on the ability of TRANCE to induce osteoclastic differentiation.

Prostaglandin E2, interleukin 1alpha, and tumor necrosis factor-alpha increase human osteoclast formation and bone resorption in vitro.

Prostaglandin E2 (PGE2) and the cytokines interleukin (IL) 1alpha and tumor necrosis factor (TNF)alpha increase bone resorption in vivo, but the effect of these agents on osteoclastic bone resorption has never been studied in an in vitro human system. Our recently described human bone marrow culture system, in which osteoclasts are generated (vitronectin and calcitonin receptor-positive cells which resorb bone), was used to study the effects of these agents. Addition of indomethacin to macrophage colony-stimulating factor (M-CSF)-treated cultures nearly abolished osteoclast parameters, indicating that prostaglandins are virtually essential for human osteoclast formation. Additionally, PGE2 dose responsively increased osteoclast numbers and bone resorption. The effects of M-CSF and PGE2 are independent, as demonstrated by unaltered PGE2 concentrations in culture supernatants in spite of the dose-responsive increase in osteoclast parameters in response to M-CSF. The generation of osteoclasts in the presence of PGE2 occurred in favor of CD 14-positive macrophage formation. IL 1alpha and TNFalpha increased osteoclast parameters in a dose-responsive manner. Maximum stimulation yielded culture supernatant levels of PGE2 approximately the same as those concentrations of exogenous PGE2 that dramatically induced osteoclast formation. This osteoclast-inducing effect was inhibited both by indomethacin and by the specific inhibitor of inducible prostaglandin G/H synthase, NS398, and this was reversed by addition of exogenous PGE2. These results demonstrate unequivocally that IL 1alpha and TNFalpha enhance human osteoclast formation and suggest that they mediate their effects through PGE2.

Inhibition of bone resorption by 17β‐estradiol in human bone marrow cultures

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17β-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17β-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17β-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17β-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17β-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17β-E2-induced inhibition of bone resorption. J. Cell. Physiol. 175:99–108, 1998. © 1998 Wiley-Liss, Inc.

Inhibition of bone resorption by 17beta-estradiol in human bone marrow cultures.

Estrogen deficiency puts individuals at risk of developing osteoporosis because it causes increased bone resorption. However, the mechanism by which this occurs is not known. We have shown, using a recently described two-phase human bone marrow culture system, that estradiol (17beta-E2) added to phase I results in inhibition of bone resorption by reducing the number of osteoclasts (identified as vitronectin receptor and/or calcitonin receptor-positive cells) formed in the cultures. The addition of 17beta-E2 in phase II was without effect, indicating that it does not interfere with the bone resorptive process. 17Beta-E2 down-regulated mRNA expression and protein synthesis of the membrane form of macrophage colony-stimulating factor (M-CSF). 17Beta-E2 did not the alter the expression of the 4.0 kb M-CSF transcript. However, it increased protein synthesis of the proteoglycan form of M-CSF, but not the 85 kDa soluble form in the same cultures. Finally, addition of M-CSF to the cultures reversed the 17beta-E2-induced inhibitory effect. These observations suggest that regulation of the synthesis of membrane-bound M-CSF plays a role in 17beta-E2-induced inhibition of bone resorption.

Calcitonin Receptors as Markers for Osteoclastic Differentiation: Correlation between Generation of Bone-Resorptive Cells and Cells that Express Calcitonin Receptors in Mouse Bone Marrow Cultures*

The osteoclast is the cell that resorbs bone. It is known to derive from hemopoietic precursors, but analysis of lineage and regulation of differentiation has been hampered by lack of a specific marker that enables identification of cells of osteoclastic phenotype. Previously used markers, such as multinuclearity, that are specific for osteoclasts in bone become less specific in culture. Uniquely among bone and bone marrow cells, osteoclasts possess abundant calcitonin (CT) receptors. We therefore tested the correlation between the generation of bone-resorptive function and the formation of CT receptor-positive cells from hemopoietic tissue in vitro. Without 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], a hormone that induces osteoclastic differentiation in vitro, bone marrow cultures showed very little bone resorption, and only small numbers of CT receptor-positive cells developed. When 1,25-(OH)2D3 was added to the cultures, CT receptor-positive cells developed within 1 day and reached a peak after 7 days. Bone resorption commenced within 2 days of hormone addition. There was a strong parallelism between the cumulative number of CT receptor-positive cells and the extent of bone resorption. The capacity of cultures to generate bone-resorptive activity and CT receptor-positive cells declined progressively when 1,25-(OH)2D3 was added to hemopoietic tissue after a 7- to 21-day hormone-free incubation period. The number of CT receptor-positive cells in these cultures correlated strongly (r = 0.96) with bone resorption. The behavior of these cultures suggests that 1,25-(OH)2D3 acts to induce terminal differentiation of osteoclast precursors present in the cultures, and that precursor cell numbers decreased with increasing time in vitro. All of the CT receptor-positive cells in control cultures and all of those seen shortly after 1,25-(OH)2D3 addition were mononuclear, despite considerable bone resorption; the majority of CT receptor-positive cells remained mononuclear throughout the incubation period. This suggests that mononuclear cells with characteristics of osteoclasts exist that are able to excavate bone. CT receptor-positive cells slightly preceded the development of bone-resorptive function, implying that CT receptors develop before the acquisition of bone-resorptive capacity by osteoclasts. Peritoneal macrophages, blood mononuclear cells, and cells of the J774 macrophage cell line failed to either resorb bone or express CT receptors, even after incubation with 1,25-(OH)2D3 for 14 days. These results show a strong and specific correlation between the generation of bone-resorptive cells and CT receptor-positive cells, and suggest that CT receptor express