Anja Nohe

The Mode of Bone Morphogenetic Protein (BMP) Receptor Oligomerization Determines Different BMP-2 Signaling Pathways

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-inducedversus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.

Molecular basis of the potential of vitamin D to prevent cancer

ABSTRACT Objective: To review current research findings in cell biology, epidemiology, preclinical, and clinical trials on the protective effects of vitamin D against the development of cancers of the breast, colon, prostate, lung, and ovary. Current recommendations for optimal vitamin D status, the movement towards revision of standards, and reflections on healthy exposure to sunlight are also reviewed. Search methodology: A literature search was conducted in April and updated in September 2007. The Medline and Web of Knowledge databases were searched for primary and review articles published between 1970 and 2007, using the search terms ‘vitamin D’, ‘calcitriol’, ‘cancer’, ‘chemoprevention’, ‘nuclear receptor’, ‘vitamin D receptor’, ‘apoptosis’, ‘cell cycle’, ‘epidemiology’, and ‘cell adhesion molecule’. Articles that focused on epidemiological, preclinical, and clinical evidence for vitamin Ds effects were selected and additional articles were obtained from reference lists of the retrieved articles. Findings: An increasing body of research supports the hypothesis that the active form of vitamin D has significant, protective effects against the development of cancer. Epidemiological studies show an inverse association between sun exposure, serum levels of 25(OH)D, and intakes of vitamin D and risk of developing and/or surviving cancer. The protective effects of vitamin D result from its role as a nuclear transcription factor that regulates cell growth, differentiation, apoptosis and a wide range of cellular mechanisms central to the development of cancer. A significant number of individuals have serum vitamin D levels lower than what appears to protect against cancer, and the research community is currently revising the guidelines for optimal health. This will lead to improved public health policies and to reduced risk of cancer. Conclusions: Research strongly supports the view that efforts to improve vitamin D status would have significant protective effects against the development of cancer. The clinical research community is currently revising recommendations for optimal serum levels and for sensible levels of sun exposure, to levels greater than previously thought. Currently, most experts in the field believe that intakes of between 1000 and 4000 IU will lead to a more healthy level of serum 25(OH)D, at approximately 75 nmol/L that will offer significant protection effects against cancers of the breast, colon, prostate, ovary, lungs, and pancreas. The first randomized trial has shown significant protection against breast cancer, and other clinical trials will follow and ultimately lead to improved public health policies and significantly fewer cancers.

Initiation of Smad-dependent and Smad-independent signaling via distinct BMP-receptor complexes.

Background: BMP-2 (bone morphogenetic protein-2) signals via two types of transmembrane serine/threonine kinase receptors (BRI and BRII), which form heteromeric complexes prior to and after ligand binding. Within a BMP-bound receptor complex, BRII transphosphorylates and activates BRI-a for further signaling. We investigated which signaling pathway is initiated by BMP-2 via preformed receptor complexes versus BMP-2-induced signaling receptor complexes.Methods: Immunofluorescence co-patching was used to study the oligomerization of receptors at the surface of live cells. Binding and chemical cross-linking of iodinated BMP-2 followed by immunoprecipitation was used to show association of receptors in the presence of ligand. Western blots with use of anti-phospho-Smad1 antibodies and reporter gene assays with use of SBE-lux were employed to show activation of the Smad pathway. Phosphorylation of p38-MAPK was shown by Western blots. Induction of alkaline phosphatase was determined by staining the cells. The cluster density of receptors was determined with use of image correlation spectroscopy.Results and Conclusion: We showed that the Smad pathway is induced by preformed receptor complexes, whereas BMP-2-induced signaling complexes result in the activation of p38-MAPK. We also found evidence that the clustering of BRI-a at the membrane is altered in the presence of BRII, suggesting that it associates with existing clusters of BRII to initiate efficient Smad signaling. These data clearly demonstrate that it is critical to fully understand receptor oligomerization in order to estimate signaling outcome for distinct receptor and ligand mutants.Clinical Relevance: The development of BMP-2 antagonists is of special importance for a number of human disorders caused by several members of the BMP/TGF-&bgr; (transforming growth factor-beta) superfamily. Since manipulation of BMP-signaling is complex, it is important to understand what influence it might have during the initiation of signaling—:i.e., the oligomerization of BMP receptors to form a signaling receptor complex. There might be cases where either the Smad or the p38 pathway should be targeted.

Dynamics and interaction of caveolin-1 isoforms with BMP-receptors

Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms α and β on the cell surface. BRIa colocalizes predominantly with the caveolin-1 α isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 β. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 α. BRII is expressed in regions enriched in caveolin-1 α and β. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 α. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 α and β. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 β isoform inhibits BMP signaling whereas the α isoform does not.

Effect of the distribution and clustering of the type I A BMP receptor (ALK3) with the type II BMP receptor on the activation of signalling pathways.

Bone morphogenetic proteins (BMPs) play an important role during embryonic development, especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. There are over 19 BMPs known in mammalians, but only three BMP-type-I receptors and three BMP-type-II receptors are known so far to mediate these responses. Previous reports provide evidence to support that oligomerisation of BMP receptors influences the activation of the downstream BMP signalling pathways, the Smad or the p38 MAPK pathway. To further explore the importance of BMP receptor clustering in signalling, image correlation spectroscopy has been used to investigate the clustering and distribution of BMP receptors at the surface of the cell membrane. Here we demonstrate that the co-expression of the BMP-type-II receptor (BRII) influences the aggregation and the distribution of the BMP-type-Ia receptor (BRIa) in COS7 cells and in A431 cells. We also demonstrate that BMP-2 stimulation of the cells leads to a rearrangement of receptor complexes at the cell surface. Using A431 cells and limb bud-derived mesenchymal cells, we show that co-expression of the BRII and a constitutive active BRIa-ca is necessary for the activation of the Smad pathway. Importantly using a kinase-inactive BRII the rearrangement of BRIa is blocked. Together, these findings suggest that rearrangement of the receptors at the cell surface prior to forming preformed ligand independent complexes plays a critical role in activation of the Smad pathway. It also suggests further that the kinase activity of BRII is needed for signalling beyond the activation of BRIa at the GS domain.

Image Correlation Spectroscopy

Membrane domains, such as caveolae and clathrin-coated pits, regulate cell signaling and protein internalization in the plasma membrane. Fluorescence imaging and microscopy provide an opportunity to determine the receptor protein dynamics of membrane microdomains. The family of image correlation spectroscopy (ICS) techniques provides powerful tools with which to measure the aggregation, clustering, and dynamics of proteins in the plasma membrane. ICS is used to calculate the cluster density and the degree of aggregation of plasma membrane proteins, whereas image cross-correlation spectroscopy (ICCS) measures the fraction of colocalization of two proteins. Dynamic image correlation spectroscopy (DICS) can be used to analyze protein dynamics on the cell surface during live-cell imaging.

Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.

Abstract Pohl, F., Hassel, S., Nohe, A., Flentje, M., Knaus, P., Sebald, W. and Koelbl, O. Radiation-Induced Suppression of the Bmp2 Signal Transduction Pathway in the Pluripotent Mesenchymal Cell Line C2C12: An In Vitro Model for Prevention of Heterotopic Ossification by Radiotherapy. Radiat. Res. 159, 345–350 (2003). Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.

Caveolin-1 isoform reorganization studied by image correlation spectroscopy

Caveolae are small, flask shaped invaginations in the cell membrane. They are thought to play a crucial role in cell signaling, endocytosis and intracellular cholesterol transport. Caveolin-1, 2 and 3 are key proteins, which are important for the formation of the invaginations on the cell surface. Caveolin-1 exists in two isoforms: caveolin-1 alpha (a) and caveolin-1 beta (beta). Little is known about the difference between these two isoforms, and less in known about their role in cell signaling. Bone morphogenetic proteins IBMPs) are a subfamily of the TGF beta superfamily and their response is mediated by serine/threonine kinase receptors. Epidermal growth factor (EGF) is known to signal through tyrosine kinase receptors of the ErbB family. Here we report on the aggregation and association of caveolin-1 isoforms with these receptors and the effect of BMP and EGF activation on caveolin-1 distribution in A431 cells. Our data, obtained by application of a family of image correlation spectroscopy tools, indicate that BMP and EGF stimulation lead to a rearrangement of the caveolin-1 isoforms on the cell surface. BMP as well as EGF stimulation leads to a rearrangement of the caveolin-1 P isoform into domains enriched in the caveolin-1 alpha isoform. We further show that about 20-30% of the caveolin-1 present at the surface of the cells co-localize with the EGF and BMP receptors. Using a reporter gene assay sensitive to the activation of the BMP pathway, we show that overexpression of caveolin-1beta inhibits signaling. Our data suggest that the two isoforms of caveolin-1 play different roles on the cell surface and that caveolae are dynamic structures.

Analyzing for co-localization of proteins at a cell membrane.

Cell-to-cell communication is mediated by molecular interactions at the surface of the cell by soluble ligands released from distant cells or by cell surface molecules on adjacent cells. These interactions lead to activation of intracellular signaling pathways that subsequently can lead to activation of specific genes. This signal transduction process controls cellular activities as diverse as proliferation, differentiation and apoptosis, so we must understand the underlying molecular events in detail in order to understand broader questions related to development, uncontrolled growth in tumors, tissue regeneration and use of stem cells to name a few. Binding of a ligand in the extracellular space to a transmembrane receptor constitutes the first crucial step for activation of a signaling pathway within the cell. This binding can either lead to oligomerization of individual receptors, to reorganization of existing clusters of receptors or to changes in the protein conformations, which in turn results in recruitment of signaling molecules in the cytoplasm. While different membrane receptors activate different downstream signaling pathways, some receptors can activate more than one pathway and a particular pathway can be activated by different receptors. It appears that these processes are regulated either by agonists and antagonists in the extracellular medium, by receptor-receptor interactions in the membrane or by a number of signaling mediators in the cytoplasm of the cell. Our work has focused on understanding how the intermolecular interactions in the membrane can control the signal transduction process: Are there specialized structures on the surface that facilitate receptor-receptor interactions? Do the receptors exist as monomers or pre-existing complexes that enhance the probability of activation? Do different receptors associate in the same domains or are there distinct organizational principles for each receptor type. In order to address these questions, we seek to develop tools that allow us to examine intermolecular interactions and reactions directly on the cell surface, particularly on live cells in culture or in tissue. This review discusses some of the approaches that are currently available and highlights some of the key advantages and disadvantages they represent with particular focus on image cross correlation spectroscopy as a relatively new quantitative tool developed by us to address some of these issues.

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.