Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4

Vascular calcification is a common complication in atherosclerosis. Bone morphogenetic protein-2 (BMP-2) plays an important role in atherosclerotic vascular calcification. The aim of this study was to determine the effect of oxidized low density lipoprotein (oxLDL) on BMP-2 protein expression in human coronary artery endothelial cells (CAECs), the roles of Toll-like receptor (TLR) 2 and TLR4 in oxLDL-induced BMP-2 expression, and the signaling pathways involved. Human CAECs were stimulated with oxLDL. The roles of TLR2 and TLR4 in oxLDL-induced BMP-2 expression were determined by pretreatment with neutralizing antibody, siRNA, and overexpression. Stimulation with oxLDL increased cellular BMP-2 protein levels in a dose-dependent manner (40-160 μg/ml). Pretreatment with neutralizing antibodies against TLR2 and TLR4 or silencing of these two receptors reduced oxLDL-induced BMP-2 expression. Overexpression of TLR2 and TLR4 enhanced the cellular BMP-2 response to oxLDL. Furthermore, oxLDL was co-localized with TLR2 and TLR4. BMP-2 expression was associated with activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK)1/2. Inhibition of NF-κB and ERK1/2 reduced BMP-2 expression whereas inhibition of p38 MAPK had no effect. In conclusion, oxLDL induces BMP-2 expression through TLR2 and TLR4 in human CAECs. The NF-κB and ERK1/2 pathways are involved in the signaling mechanism. These findings underscore an important role for TLR2 and TLR4 in mediating the BMP-2 response to oxLDL in human CAECs and indicate that these two immunoreceptors contribute to the mechanisms underlying atherosclerotic vascular calcification.     

Bone Morphogenetic Proteins Signal Via SMAD and Mitogen-activated Protein (MAP) Kinase Pathways at Distinct Times during Osteoclastogenesis

To investigate the role of bone morphogenetic protein (BMP) signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRII^fl/fl;lysM-Cre mouse strain. Conditional knock-out (cKO) mice are osteopetrotic when compared with WT controls due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M-CSF and receptor activator of NF-κB (RANK) ligand. We also show that BMP noncanonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre-fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Different requirements for proteolytic processing of bone morphogenetic protein 5/6/7/8 ligands in Drosophila melanogaster

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.     

Lung tumor-associated osteoblast-derived bone morphogenetic protein-2 increased epithelial-to-mesenchymal transition of cancer by Runx2/Snail signaling pathway

Bone is a frequent target of lung cancer metastasis and is associated with significant morbidity and a dismal prognosis. Interaction between cancer cells and the bone microenvironment causes a vicious cycle of tumor progression and bone destruction. This study analyzed the soluble factors secreted by lung tumor-associated osteoblast (TAOB), which are responsible for increasing cancer progression. The addition of bone morphogenetic protein-2 (BMP-2), present in large amounts in TAOB conditioned medium (TAOB-CM) and lung cancer patient sera, mimicked the inductive effect of TAOB-CM on lung cancer migration, invasion, and epithelial-to-mesenchymal transition. In contrast, inhibition of BMP by noggin decreases the inductive properties of TAOB-CM and lung cancer patient sera on cancer progression. Induction of lung cancer migration by BMP-2 is associated with increased ERK and p38 activation and the up-regulation of Runx2 and Snail. Blocking ERK and p38 by a specific inhibitor significantly decreases cancer cell migration by inhibiting Runx2 up-regulation and subsequently attenuating the expression of Snail. Enhancement of Runx2 facilitates Rux2 to recruit p300, which in turn enhances histone acetylation, increases Snail expression, and decreases E-cadherin. Furthermore, inhibiting Runx2 by siRNA also suppresses BMP-2-induced Snail up-regulation and cell migration. Our findings provide novel evidence that inhibition of BMP-2 or BMP-2-mediated MAPK/Runx2/Snail signaling is an attractive therapeutic target for osteolytic bone metastases in lung cancer patients.     

Novel contact-dependent bone morphogenetic protein (BMP) signaling mediated by heparan sulfate proteoglycans

We previously proposed a model that DALLY, a Drosophila glypican, acts as a trans co-receptor to regulate BMP signaling in the germ line stem cell niche. To investigate the molecular mechanisms of contact-dependent BMP signaling, we developed novel in vitro assay systems to monitor trans signaling using Drosophila S2 cells. Using immunoblot-based as well as single-cell assay systems, we present evidence that Drosophila glypicans indeed enhance BMP signaling in trans in a contact-dependent manner in vitro. Our analysis showed that heparan sulfate modification is required for the trans co-receptor activity of DALLY. Two BMP-like molecules, Decapentaplegic (DPP) and Glass bottom boat, can mediate trans signaling through a heparan sulfate proteoglycan co-receptor in S2 cells. The in vitro systems reflect the molecular characteristics of heparan sulfate proteoglycan functions observed previously in vivo, such as ligand specificity and biphasic activity dependent on the ligand dosage. In addition, experiments using a DALLY-coated surface suggested that DALLY regulates DPP signaling in trans by its effect on the stability of DPP protein on the surface of the contacting cells. Our findings provide the molecular foundation for novel contact-dependent signaling, which defines the physical space of the stem cell niche in vivo.     

Syk regulates multiple signaling pathways leading to CX3CL1 chemotaxis in macrophages

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.     

Gadd45b Mediates Fas-induced Apoptosis by Enhancing the Interaction between p38 and Retinoblastoma Tumor Suppressor

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.     

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

Background  

Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells.     

The p38β Mitogen-activated Protein Kinase Possesses an Intrinsic Autophosphorylation Activity,Generated by a Short Region Composed of the α-G Helix and MAPK Insert

Protein kinases are regulated by a large number of mechanisms that vary from one kinase to another. However, a fundamental activation mechanism shared by all protein kinases is phosphorylation of a conserved activation loop threonine residue. This is achieved in many cases via autophosphorylation. The mechanism and structural basis for autophosphorylation are not clear and are in fact enigmatic because this phosphorylation occurs when the kinase is in its inactive conformation. Unlike most protein kinases, MAP kinases are not commonly activated by autophosphorylation but rather by MEK-dependent phosphorylation. Here we show that p38β, a p38 isoform that is almost identical to p38α, is exceptional and spontaneously autoactivates by autophosphorylation. We identified a 13-residue-long region composed of part of the αG-helix and the MAPK insert that triggers the intrinsic autophosphorylation activity of p38β. When inserted into p38α, this fragment renders it spontaneously active in vitro and in mammalian cells. We further found that an interaction between the N terminus and a particular region of the C-terminal extension suppresses the intrinsic autophosphorylation of p38β in mammalian cells. Thus, this study identified the structural motif responsible for the unique autophosphorylation capability of p38β and the motif inhibiting this activity in living cells. It shows that the MAPK insert and C-terminal extension, structural motifs that are unique to MAPKs, play a critical role in controlling autophosphorylation.