Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75^NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75^NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75^NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75^NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75^NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75^NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75^NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.     

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.     

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts

Interleukin-17 (IL-17) is a cytokine secreted primarily by T_H-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE₂ production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE₂, in osteoblasts.     

Sonic hedgehog is involved in osteoblast differentiation by cooperating with BMP-2

The roles of Sonic hedgehog (Shh) and Bone morphogenetic protein-2 (Bmp-2) in osteoblast differentiation were investigated using in vitro cell systems. Recombinant amino-terminal portion of SHH (rSHH-N) dose dependently stimulated ALP activity in C3H10T1/2 and MC3T3-E1 cells. rSHH-N induced expression of Osteocalcin mRNA in C3H10T1/2 cells. A soluble form of the receptor for type IA BMP receptor antagonized rSHH-N-induced ALP activity in C3H10T1/2 and MC3T3-E1 cells, indicating that BMPs are involved in SHH-induced osteoblast differentiation. Simultaneous supplement with rSHH-N and BMP-2 synergistically induced ALP activity and expression of Osteocalcin mRNA in C3H10T1/2 cells. Pretreatment with rSHH-N for 6 h enhanced the response to BMP-2 by increasing ALP activity in C3H10T1/2 and MC3T3-E1 cells. Stimulatory effects of rSHH-N and additive effects with rSHH-N and BMP-2 on ALP activity were also observed in mouse primary osteoblastic cells. Transplantation of BMP-2 (1 microg) into muscle of mice induced formation of ectopic bone, whereas transplantation of r-SHH-N (1-5 microg) failed to generate it. These results indicate that Shh plays important roles in osteoblast differentiation by cooperating with BMP.     

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of Escherichia coli

Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE(2) and NO assay showed that LIPUS could enhance PGE(2) and NO secretion from MLO-Y4 cells at all time points within 24h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE(2) from osteocytes may play a role in this effect.     

Role of Src-specific phosphorylation site on focal adhesion kinase for senescence-associated apoptosis resistance

A decreased apoptotic response toward noxious stress is an issuing characteristic of the aging phenotype. Hydrogen peroxide or staurosporine induced apoptosis readily in young cells but not in senescent cells. We showed that focal adhesion kinase (FAK) expression and its phosphorylation at Tyr³⁹⁷, autophosphorylation site for focal adhesion formation, and Tyr⁵⁷⁷, Src-dependent phosphorylation site, were both increased in senescent cells. Moreover, FAK was inactivated proteolytically by apoptotic stimuli in young cells, but not in senescent cells. In addition, senescent cells whose FAK expression was downregulated by siRNA showed the increased level of apoptosis by staurosporine treatment via caspase-3 activation but not by hydrogen peroxide treatment. Interestingly dephosphorylation at Tyr⁵⁷⁷ of FAK by PP2 treatment, Src-family kinase inhibitor, induced the apoptosis by staurosporine in senescent cells but dephosphorylation at Tyr³⁹⁷ by downregulation of caveolin-1 was not affected. These data suggest that FAK might differently regulate apoptosis and focal adhesion formation through site-specific tyrosine phosphorylation in senescent cells. Both authors contributed equally to this work.     

Involvement of ligand occupancy in Insulin-like growth factor-I (IGF-I) induced cell growth in osteoblast like MC3T3-E1 cells

Growth factors and matrix proteins regulate the proliferation and differentiation of osteoblasts. The insulin-like growth factor (IGF) system comprises IGF-I, IGF-II, and six high-affinity IGF-binding proteins (IGFBPs). IGFs stimulate cell growth in many types of tissue; IGF-binding proteins regulate cellular actions and can affect cell growth. IGF-I is involved in differentiation, proliferation, and matrix formation in osteoblasts; IGFBP-5 is associated with the extracellular matrix (ECM) and can potentiate the actions of IGF-I. We investigated the effect of ECM proteins on the responses of MC3T3-E1 osteoblast cells to IGF-I and IGFBP-5. In addition, because extracellular signal-regulated kinases 1 and 2 (Erk 1/2) affect cell growth, we evaluated the effects of IGFBP-5 on Erk 1/2 phosphorylation in MC3T3-E1 cells. IGF-I caused an increase in IGFBP-5 expression in cultured MC3T3-E1 cells, and IGF-I plus IGFBP-5 significantly increased cell growth. Likewise, the addition of IGF-I and IGFBP-5 to cultured MC3T3-E1 cells increased the synthesis of the ECM proteins osteopontin (OPN) and thrombospondin-1 (TSP-1), which can bind to alphaVbeta3 integrin receptors on the cell surface. By contrast, the addition of an antibody against ECM proteins inhibited the effects of OPN and TSP-1 on IGFBP-5 expression. The stimulatory effect of IGFBP-5 was mediated via Erk 1/2 activation. These data suggest that IGFBP-5 regulates Erk 1/2 phosphorylation in cultured MC3T3-E1 cells via ECM proteins that may ultimately stimulate the growth of osteoblasts. We determined whether occupation of the alphaVbeta3 integrin receptor affects IGF-I receptor (IGF-IR)-mediated signaling and function in MC3T3-E1 osteoblast cells. Occupation of the alphaVbeta3 integrin receptor with ECM proteins induced IGF-I-stimulated IGF-IR phosphorylation. Conversely, in the presence of the alphaVbeta3-specific disintegrin echistatin, IGF-I-stimulated IGF-IR activation was inhibited. IGF-I-stimulated IGF-IR phosphorylation was accompanied by IRS-1 phosphorylation and MAPK activation. However, these effects were attenuated by echistatin. Thus, occupancy of the alphaVbeta3 disintegrin receptor modulates IGF-I-induced IGF-IR activation and IGF-IR-mediated function in MC 3T3-E1 osteoblasts.     

Icariin ameliorates estrogen-deficiency induced bone loss by enhancing IGF-I signaling via its crosstalk with non-genomic ERα signaling

BackgroundRapid, non-genomic estrogen receptor (ER) signaling plays an integral role in mediating the tissue selective properties of ER modulators. Icariin, a bone bioactive flavonoid, has been reported to selectively activate non-genomic ERα signaling in in vitro and in vivo studies.PurposeThe mechanisms underlying the estrogen-like bone protective effects of icariin are not fully understood, especially those that are related to insulin-like growth factor I (IGF-1) signaling. The bone protective effects of icariin were investigated in female mature ovariectomized (OVX) rats and the signaling of IGF-IR- ERα cross-talk was determined in osteoblastic cells.Study design and methodsIcariin at 3 different dosages (50, 500 and 3000 ppm) were orally administrated to rats for 3 months through daily intake of phytoestrogen-free animal diets containing icariin. Bone marrow stromal cells (BMSCs) and osteoclast precursors from femurs were harvested for experiments and RNA-sequencing. The interactions between IGF-IR and non-genomic ERα signaling were examined in pre-osteoblastic MC3T3-E1 cells and mature osteoblasts differentiated from BMSCs.ResultsOur results show that chronic administration of icariin to OVX rats significantly protected them against bone loss at the long bone and lumbar spine without inducing any uterotrophic effects. Ex vivo studies using BMSCs and osteoclast precursors confirmed the stimulatory effects of icariin on osteoblastogenesis and its inhibitory effects on osteoclastogenesis, respectively. RNA-sequencing analysis of mRNA from BMSCs revealed that icariin at 500 ppm significantly altered IGF-1 signaling as well as PI3K-Akt pathways. Our results demonstrated for the first time the rapid induction of interactions between IGF-IR and ERα as well as IGF-IR signaling and the downstream Akt phosphorylation by icariin in MC3T3-E1 cells. The activation of ERα and Akt phosphorylation by icariin in MC3T3-E1 cells and the osteogenic effects of icariin on ALP activity in mature osteoblasts were shown to be IGF-IR-dependent.ConclusionOur findings reveal that icariin activates both ERα and Akt via enhancing rapid induction of IGF-1 signaling in osteoblastic cells for osteogenesis and might be regarded as a novel pathway-selective phytoestrogen for management of postmenopausal osteoporosis.     

The Role of Sonic Hedgehog Signaling in Osteoclastogenesis and Jaw Bone Destruction

Sonic hedgehog (SHH) and its signaling have been identified in several human cancers, and increased levels of its expression appear to correlate with disease progression and metastasis. However, the role of SHH in bone destruction associated with oral squamous cell carcinomas is still unclear. In this study we analyzed SHH expression and the role played by SHH signaling in gingival carcinoma-induced jawbone destruction. From an analysis of surgically resected lower gingival squamous cell carcinoma mandible samples, we found that SHH was highly expressed in tumor cells that had invaded the bone matrix. On the other hand, the hedgehog receptor Patched and the signaling molecule Gli-2 were highly expressed in the osteoclasts and the progenitor cells. SHH stimulated osteoclast formation and pit formation in the presence of the receptor activator for nuclear factor-κB ligand (RANKL) in CD11b⁺ mouse bone marrow cells. SHH upregulated phosphorylation of ERK1/2 and p38 MAPK, NFATc1, tartrate-resistant acid phosphatase (TRAP), and Cathepsin K expression in RAW264.7 cells. Our results suggest that tumor-derived SHH stimulated the osteoclast formation and bone resorption in the tumor jawbone microenvironment.     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.     

牙源性间充质干细胞对成骨前体细胞成骨分化的影响

目的:探讨牙源性间充质干细胞对成骨前体细胞成骨分化的影响.方法:将小鼠成骨前体细胞MC3T3-El分为两组,观察组为牙源性间充质干细胞与MC3T3-E1细胞共培养,对照组为单一MC3T3-E1细胞培养.采用CCK-8法检测细胞增殖水平,采用酶联免疫法检测碱性磷酸酶(Alkaline phosphatase,ALP)活性...     

IGFBP-4 degradation by pregnancy-associated plasma protein-A in MC3T3 osteoblasts

Insulin-like growth factor (IGF) signaling is critical for osteoblast development and IGF binding protein (IGFBP)-4 is one of the principle IGFBPs expressed by osteoblasts. Release of bound IGF via proteolytic degradation of IGFBP-4 is likely to be critical for osteoblast development. We have investigated whether IGF-sensitive, IGFBP-4 degradation in mouse MC3T3-E1 osteoblasts is due to the metzincin pregnancy-associated plasma protein (PAPP)-A. Degradation of IGFBP-4 by PAPP-A or MC3T3-E1 conditioned medium was enhanced by IGF-II but inhibited by mutation of basic residues at or near the PAPP-A cleavage site in IGFBP-4. Furthermore, immunodepletion of PAPP-A from MC3T3-E1 conditioned medium abolished IGFBP-4 degradation. We also found that PAPP-A messenger RNA was expressed throughout differentiation of MC3T3-E1 cells. These results demonstrate for the first time that PAPP-A is the IGFBP-4 protease in MC3T3-E1 cells, a widely used model for osteoblast development, and that PAPP-A may regulate IGF release throughout osteoblast differentiation.     

Reciprocal gene expression of osteoclastogenesis inhibitory factor and osteoclast differentiation factor regulates osteoclast formation

Osteoblasts/stromal cells support the formation of osteoclast-like cells (OCL) from osteoclast progenitor cells via expressing a membrane-associated protein, osteoclast differentiation factor (ODF), in the presence of osteotropic factors, whereas the cells secrete a substantial amount of osteoclastogenesis inhibitory factor (OCIF) in the unstimulated state. There are both OCL formation-supporting and the nonsupporting cell lines in osteoblasts/stromal cell lineages. The mechanism that divides osteoblasts/stromal cell lines into the two types is not known. The present study reports that OCL formation-supporting cell line ST2 showed a greatly increased level of ODF mRNA, whereas their OCIF mRNA was drastically diminished in the presence of 1alpha, 25(OH)2-dihydroxyvitamin D3 or prostaglandin E2. In contrast, MC3T3-E1 cells lacking OCL formation-supporting ability did not show a decrease in OCIF mRNA in response to the factors, despite a similar increase in ODF mRNA as ST2 cells. However, inactivated MC3T3-E1 cells secreting nothing supported OCL formation in coculture with human promyelocytic cells, HL60. On the contrary, ST2 cells did not support OCL formation from HL60 cells when cocultured in medium conditioned by 1alpha, 25(OH)2 vitamin D3-treated MC3T3-E1. These findings indicate that reciprocal gene expression of ODF and OCIF in osteoblasts/stromal cells is essential for supporting OCL formation.