Controlling of bone morphogenetic protein signaling

Bone morphogenetic proteins (BMPs) are multifunctional growth factors and play crucial roles during embryonic development, skeletal development, and cell fate determination. Their signals are transduced from cell membrane to the nucleus through intracellular signaling mediators. At present, different signaling pathways have been identified, and elaborate of network of regulators involved in the signaling control. The aim of the present review is to describe the recent understanding of BMPs signaling with emphasis on the regulation of its signal transduction at extracellular level, intracellular level, Smad-interacting factors in the nucleus, and Smad-independent signaling pathways, respectively.     

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.     

Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction.     

Role of RGM coreceptors in bone morphogenetic protein signaling

Background

The repulsive guidance molecule (RGM) proteins, originally discovered for their roles in neuronal development, have been recently identified as co-receptors in the bone morphogenetic protein (BMP) signaling pathway. BMPs are members of the TGFβ superfamily of signaling cytokines, and serve to regulate many aspects of cellular growth and differentiation.

Results

Here, we investigate whether RGMa, RGMb, and RGMc play required roles in BMP and TGFβ signaling in the mouse myoblast C2C12 cell line. These cells are responsive to BMPs and are frequently used to study BMP/TGFβ signaling pathways. Using siRNA reagents to specifically knock down each RGM protein, we show that the RGM co-receptors are required for significant BMP signaling as reported by two cell-based BMP activity assays: endogenous alkaline phosphatase activity and a luciferase-based BMP reporter assay. Similar cell-based assays using a TGFβ-induced luciferase reporter show that the RGM co-receptors are not required for TGFβ signaling. The binding interaction of each RGM co-receptor to each of BMP2 and BMP12 is observed and quantified, and equilibrium dissociation constants in the low nanomolar range are reported.

Conclusion

Our results demonstrate that the RGMs play a significant role in BMP signaling and reveal that these molecules cannot functionally compensate for one another.     

Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling

Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh-/- phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh-/- mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh-/- calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.     

New intracellular components of bone morphogenetic protein/Smad signaling cascades

Bone morphogenetic proteins (BMPs) regulate many processes in the embryo, including cell type specification, patterning, apoptosis, and epithelial-mesenchymal interaction. They also act in soft and hard tissues in adult life. Their signals are transduced from the plasma membrane to the nucleus through a limited number of Smad proteins. The list of Smad-interacting proteins is however growing and it is clear that these partners determine the outcome of the signal. We summarize the present status in BMP/Smad signaling, with emphasis on recently identified Smad partners and how these proteins may cooperate in the regulation of the expression of BMP target genes.     

Constitutively active homo-oligomeric angiotensin II type 2 receptor induces cell signaling independent of receptor conformation and ligand stimulation

Members of the G-protein-coupled receptor superfamily (GPCRs) undergo homo- and/or hetero-oligomerization to induce cell signaling. Although some of these show constitutive activation, it is not clear how such GPCRs undergo homo-oligomerization with transmembrane helix movement. We previously reported that angiotensin II (Ang II) type 2 (AT(2)) receptor, a GPCR, showed constitutive activation and induced apoptosis independent of its ligand, Ang II. In the present study, we analyzed the translocation and oligomerization of the AT(2) receptor with transmembrane movement when the receptor induces cell signaling. Constitutively active homo-oligomerization, which was due to disulfide bonding between Cys(35) in one AT(2) receptor and Cys(290) in another AT(2) receptor, was localized in the cell membrane without Ang II stimulation and induced apoptosis without changes in receptor conformation. These results provide the direct evidence that the constitutively active homo-oligomeric GPCRs by intermolecular interaction in two extracellular loops is translocated to the cell membrane and induces cell signaling independent of receptor conformation and ligand stimulation.     

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Type I collagen induces expression of bone morphogenetic protein receptor type II

The extracellular matrix regulates many fundamental cellular processes such as proliferation, migration, and differentiation. Among the ECM components, type I collagen induces endothelial tube formation in vitro. By analysing genes participating in this event, the bone morphogenetic protein receptor-II (BMPR-II) was detected to be upregulated in cells cultured on or within fibrillar type I collagen. Furthermore, the basement membrane type IV collagen or amorphous type I collagen did not show an induction of BMPR-II. Addition of the BMPR-II specific ligands, BMP2 and BMP4, in the culture medium of the endothelial cells seeded on type I collagen increased [(3)H]-thymidine incorporation into cellular DNA, indicating that endothelial cells were able to form a functional receptor. In addition, in the chick chorioallantoic membrane (CAM), an in vivo angiogenesis model, BMPR-II and BMPR-I were upregulated in the growing phase and ceased in the mature CAM.     

Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-beta type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.