Caveolae regulate Smad signaling as verified by novel imaging and system biology approaches.

The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin-coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, and sucrose density centrifugation followed by Western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad-dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor-ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied.

Initiation of BMP2 signaling in domains on the plasma membrane.

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for cell fate determination. It directs the differentiation of mesenchymal stem cells into osteoblasts, chondrocytes, adipocytes, and myocytes. Initiation of BMP2 signaling pathways occurs at the cell surface through type I and type II serine/threonine kinases housed in specific membrane domains such as caveolae enriched in the caveolin-1 beta isoform (CAV1ß, caveolae) and clathrin-coated pits (CCPs). In order for BMP2 to initiate Smad signaling it must bind to its receptors on the plasma membrane resulting in the phosphorylation of the BMP type Ia receptor (BMPRIa) followed by activation of Smad signaling. The current model suggests that the canonical BMP signaling pathway, Smad, occurs in CCPs. However, several recent studies suggested Smad signaling may occur outside of CCPs. Here, we determined; (i) The location of BMP2 binding to receptors localized in caveolae, CCPs, or outside of these domains using AFM and confocal microscopy. (ii) The location of phosphorylation of BMPRIa on the plasma membrane using membrane fractionation, and (iii) the effect of down regulation of caveolae on Smad signaling. Our data indicate that BMP2 binds with highest force to BMP receptors (BMPRs) localized in caveolae. BMPRIa is phosphorylated in caveolae and the disruption of caveolae-inhibited Smad signaling in the presence of BMP2. This suggests caveolae are necessary for the initiation of Smad signaling. We propose an extension of the current model of BMP2 signaling, in which the initiation of Smad signaling is mediated by BMPRs in caveolae.

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype.

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin-1 alpha and Caveolin-1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H-1-12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re-localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H-1-12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H-1-12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2-induced Smad signaling in BMSCs isolated from B6.C3H-1-12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H-1-12 congenic and (ii) contributes to increase BMD of the B6.C3H-1-12 compared to the C57BL/6J control mice.

Design of 1,25 dihydroxyvitamin D3 coupled quantum dots, a novel imaging tool.

1,25 dihydroxyvitamin D3 (Calcitriol), one of the active forms of Vitamin D, plays a vital role not only in calcium absorption but also during neuromuscular function and regulation of inflammation. Epidemiological studies suggest a preventive effect of Calcitriol in breast, colon and prostate cancer, however high concentrations of Calcitriol are necessary. Therefore targeted biologically active probes must be designed to determine Calcitriol distribution and dynamics in vitro and in vivo. Our Calcitriol probe remained stable over 2 days at 37 degrees C. When added to live C2C12 cells, the Calcitriol probe can be seen entering the nucleus within 2 hours and the probe activated the expression of the Vitamin D Response Element (VDRE), one of the major transcription elements. The Calcitriol probe provides a novel imaging tool that can be used to view Calcitriol localization and dynamics.

FRET reveals novel protein-receptor interaction of bone morphogenetic proteins receptors and adaptor protein 2 at the cell surface.

Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 beta-isoform and caveolae of the caveolin-1 alpha/beta-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.

CK2.1, a novel peptide, induces articular cartilage formation in vivo.

Bone morphogenetic protein 2 regulates chondrogenesis and cartilage formation. However, it also induces chondrocyte hypertrophy and cartilage matrix degradation. We recently designed three peptides CK2.1, CK2.2, and CK2.3 that activate the BMP signaling pathways by releasing casein kinase II (CK2) from distinct sites at the bone morphogenetic protein receptor type Ia (BMPRIa). Since BMP2 is a major regulator of chondrogenesis and the peptides activated BMP signaling in a similar way, we evaluated the effect of these peptides on chondrogenesis and cartilage formation. C3H10T1/2 cells were stimulated with CK2.1, CK2.2, and CK2.3 and evaluated for the chondrogenic and osteogenic potential. For chondrogenesis, Alcian blue staining was performed. Additionally, collagen types II and X expression was measured. For osteogenesis, osteocalcin and von Kossa staining were performed. From the three peptides, CK2.1 was the most promising peptide to induce chondrogenesis but not osteogenesis. To investigate the effect of CK2.1 on articular cartilage formation in vivo, we injected CK2.1 into the tail vein of mice. Injection of CK2.1 into the tail vein of mice led to increased articular cartilage formation but not BMD. In sharp contrast, injection of BMP2 led to increased BMD and expression of collagen type X, a marker of chondrocyte hypertrophy. MMP13 expression was unchanged. Our study demonstrates that CK2.1 drives chondrogenesis and cartilage formation without induction of chondrocyte hypertrophy. Peptide CK2.1 may, therefore, be a valuable therapeutic for cartilage degenerative diseases. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:876-885, 2017.

An Improved Immunostaining and Imaging Methodology to Determine Cell and Protein Distributions within the Bone Environment.

Bone is a dynamic tissue that undergoes multiple changes throughout its lifetime. Its maintenance requires a tight regulation between the cells embedded within the bone matrix, and an imbalance among these cells may lead to bone diseases such as osteoporosis. Identifying cell populations and their proteins within bone is necessary for understanding bone biology. Immunolabeling is one approach used to visualize proteins in tissues. Efficient immunolabeling of bone samples often requires decalcification, which may lead to changes in the structural morphology of the bone. Recently, methyl-methacrylate embedding of non-decalcified tissue followed by heat-induced antigen retrieval has been used to process bone sections for immunolabeling. However, this technique is applicable for bone slices below 50-µm thickness while fixed on slides. Additionally, enhancing epitope exposure for immunolabeling is still a challenge. Moreover, imaging bone cells within the bone environment using standard confocal microscopy is difficult. Here we demonstrate for the first time an improved methodology for immunolabeling non-decalcified bone using a testicular hyaluronidase enzyme-based antigen retrieval technique followed by two-photon fluorescence laser microscopy (TPLM) imaging. This procedure allowed us to image key intracellular proteins in bone cells while preserving the structural morphology of the cells and the bone.

Systemic injection of CK2.3, a novel peptide acting downstream of bone morphogenetic protein receptor BMPRIa, leads to increased trabecular bone mass.

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal-related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.

Synthesis and Characterization of L-Lysine Conjugated Silver Nanoparticles Smaller Than 10 nM.

A rapid and convenient batch method for synthesizing lysine-conjugated silver nanoparticles of approximately 5 nm of size was developed. Nanoparticles of size less than 100 nm exhibit significant medical potential. L-Lysine demonstrates potential for therapeutic applications and silver nanoparticles are an optimal choice for drug delivery because of its intrinsic anti-platelet, anti-bacterial and anti-inflammatory capabilities. Current synthesis protocols for Lysine-capped particles under 10 nm are time consuming and tedious and allow only for the sythesis of small quantities of particles. The synthesis of Lysin-capped silver nanoparticles was based on the reaction in which AgNO3 was reduced by excess NaBH4. L-Lysine, a known essential amino acid, served as the capping agent to minimize initial aggregation. The particles were then separated by size chromatography. Capping occurred through the amide bond on L-Lysine as determined by FT-IR. The conjugation of the particle to the amide bond is important, since this leaves the amino group of Lysine open to further modifications. The particles were further characterized in regards to their shape, size and stability. Finally we demonstrated that the synthesized particles exhibit limited to no toxicity in cells, using HEK 293 cell line as a model system. Our sythesis protocol can be successfully used for scale-up and synthesis of high quantities of nanoparticles.

Development of Physiologically Based Pharmacokinetic Model (PBPK) of BMP2 in Mice.

Bone Morphogenetic protein 2 holds great promise for potential applications in the clinic. It is a potent growth factor for the use in the cervical spine surgery (FDA approved 2002) and has been marketed as "Infuse" for treating open tibial shaft fractures (FDA approved 2004). However, its use is limited by several significant side effects that maybe due to its potency and effect on different stem cell populations in the spine. BMP2 is expressed throughout the human body in several tissues and at a very high concentration in the blood. BMP receptors, especially BMP receptor type Ia, is ubiquitously expressed in most tissues. Currently, it is difficult to determine how BMP2 is physiologically distributed in mice or humans and no quantitative models are available. A Physiologically-Based Pharmaco-Kinetic (PBPK) model has been developed to determine steady-state distribution of BMP2 in mice. The multi-compartmental PBPK model represents relevant organ/tissues with physiological accuracy. The organs/tissue compartments chosen were brain, lung, heart, liver, pancreas, kidney, uterus, bone and fat. A blood compartment maintained connectivity among the various organs. Four processes characterized the change in the concentration of the protein in every compartment: blood flow in, blood flow out, protein turnover and receptor binding in the organ. The unique aspects of the model are the determination of elimination using receptor kinetics and generation using protein turnover. The model also predicts steady state concentrations of BMP2 in tissues in mice and may be used for possible scale-up of dosage regimens in humans.

1,25 Dihydroxyvitamin D3 uptake is localized at caveolae and requires caveolar function.

Vitamin D3 is an essential vitamin that has been extensively studied due to its potential role as therapeutic for many diseases, including breast cancer. Previous research has indicated that calcitriol, the active form of Vitamin D3 has a negative effect on the metastatic ability of Inflammatory Breast Cancer (IBC) cells however the mechanism is not fully understood. The effect of calcitriol on IBC cells starting from cellular uptake must be investigated in order to understand these therapeutic effects. Calcitriol bound Quantum Dots (CalQDs) are a novel nanoparticle conjugated probe that can be used to directly examine the distribution, uptake, and signaling of calcitriol in live cells. Therefore we used these conjugated probes to directly investigate the uptake of calcitriol into live IBC cells. Interestingly, calcitriol uptake was observed to decrease when caveolae mediated endocytosis is disrupted. A luciferase assay confirmed that caveolae function is necessary; since calcitriol mediated activity decreases when caveolae mediated endocytosis is disrupted in IBC cells. In vitro examination of the localization of the probe indicated colocalization between caveolae and CalQDs. Additionally, Vitamin D Receptor (VDR) colocalization was observed with caveolae and calcitriol. This study demonstrates that in IBC cells calcitriol enters cells via caveolae mediated endocytosis and that caveolae are required for calcitriol to be uptaken at the increased rate.

Altered plasma membrane dynamics of bone morphogenetic protein receptor type Ia in a low bone mass mouse model.

Bone morphogenetic proteins (BMPs) are growth factors that initiate differentiation of bone marrow stromal cells to osteoblasts and adipocytes, yet the mechanism that decides which lineage the cell will follow is unknown. BMP2 is linked to the development of osteoporosis and variants of BMP2 gene have been reported to increase the development of osteoporosis. Intracellular signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. The BMP type I a receptor (BMPRIa) is linked to osteogenesis and bone mineral density (BMD). BMPRs are localized to caveolae enriched with Caveolin1 alpha/beta and Caveolin beta isoforms to facilitate signaling. BMP2 binding to caveolae was recently found to be crucial for the initiation of the Smad signaling pathway. Here we determined the role of BMP receptor localization within caveolae isoforms and aggregation of caveolae as well as BMPRIa in bone marrow stromal cells (BMSCs) on bone mineral density using the B6.C3H-6T as a model system. The B6.C3H-6T is a congenic mouse with decreased bone mineral density (BMD) with increased marrow adipocytes and decreased osteoprogenitor proliferation. C57BL/6J mice served as controls since only a segment of Chr6 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Family of image correlation spectroscopy was used to analyze receptor cluster density and co-localization of BMPRIa and caveolae. It was previously shown that BMP2 stimulation results in an aggregation of caveolae and BMPRIa. Additionally, BMSCs isolated from the B6.C3H-6T mice showed a dispersion of caveolae domains compared to C57BL/6J. The aggregation of BMPRIa that is necessary for signaling to occur was inhibited in BMSCs isolated from B6.C3H-6T. Additionally, we analyzed the co-localization of BMPRIa with caveolin-1 isoforms. There was increased percentage of BMPRIa co-localization with caveolae compared to C57BL/6J. BMP2 stimulation had no effect on the colocalization of BMPRIa with caveolin-1. Disrupting caveolae initiated Smad signaling in the isolated BMSCs from B6.C3H-6T. These data suggest that in congenic 6T mice BMP receptors aggregation is inhibited causing an inhibition of signaling and reduced bone mass.

Casein kinase 2 regulates in vivo bone formation through its interaction with bone morphogenetic protein receptor type Ia.

Approximately 7.9 million fractures occur annually in the United States with 5-10% of these resulting in delayed or impaired healing. Nearly half of the trauma cost of $56 billion per year is used for the treatment of fractures. More importantly, fracture results in a substantial reduction in the quality of life. New approaches and therapies are needed to enhance fracture healing. Only a limited number of treatments are available including bone grafting, allogeneic and autologous bone marrow transplantation, and bone morphogenetic protein (BMP). We previously identified Protein Kinase CK2 to interact with BMP receptor type Ia (BMPRIa) and as a key protein for signal activation. Peptides approximately 30 AA were developed that mimicked BMP2 action in vitro by blocking this interaction. In this paper we extended our studies to investigate if the most promising peptide could induce in vivo bone formation in mice and to elucidate this mechanism of action. The CK2 blocking peptide activated the Wnt pathway. To identify the optimal peptide concentration and peptide concentration curves for mineralization studies were performed. We designed BMPRIa mutants with a point mutation in the CK2 phosphorylation site to establish a specific effect. Mineralization was initiated with the overexpression of the BMPRIa mutants indicating CK2 is a negative regulatory protein for osteoblast differentiation. Osteoclast differentiation and activity was decreased with the CK2 blocking peptide. Further, subcutaneous calvarial bone injections of a CK2 blocking peptide increased bone area, areal bone mineral density, and bone growth. These results indicate CK2 is crucial for osteoblast differentiation and could be a target for future therapeutics of fracture healing.

Image correlation spectroscopy to define membrane dynamics.

Fluorescent imaging techniques are powerful tools that aid in studying protein dynamics and membrane domains and allow for the visualization and data collection of such structures as caveolae and clathrin-coated pits, key players in the regulation of cell communication and signaling. The family of image correlation spectroscopy (FICS) provides a unique way to determine details about aggregation, clustering, and dynamics of proteins on the plasma membrane. FICS consists of many imaging techniques which we will focus on including image correlation spectroscopy, image cross-correlation spectroscopy and dynamic image correlation spectroscopy. Image correlation spectroscopy is a tool used to calculate the cluster density, which is the average number of clusters per unit area along with data to determine the degree of aggregation of plasma membrane proteins. Image cross-correlation spectroscopy measures the colocalization of proteins of interest. Dynamic image correlation spectroscopy can be used to analyze protein aggregate dynamics on the cell surface during live-cell imaging in the millisecond to second range.