Corinne E. Camalier

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts.

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast‐specific factors, ion channels, and signal transduction molecules, such as 14‐3‐3 family members and Rab‐related proteins. To compare the proteome of MV with that of the ECM we conducted a large‐scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non‐redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization‐regulating vesicles. J. Cell. Physiol. 210: 325–335, 2007.

A Combined Proteome and Microarray Investigation of Inorganic Phosphate-induced Pre-osteoblast Cells

Inorganic phosphate, which is generated during osteoblast differentiation and mineralization, has recently been identified as an important signaling molecule capable of altering signal transduction pathways and gene expression. A large scale quantitative proteomic investigation of pre-osteoblasts stimulated with inorganic phosphate for 24 h resulted in the identification of 2501 proteins, of which 410 (16%) had an altered abundance ratio of greater than or equal to 1.75-fold, either up or down, revealing both novel and previously defined osteoblast-regulated proteins. A pathway/function analysis of these proteins revealed an increase in cell cycle and proliferation that was subsequently verified by conventional biochemical means. To further analyze the mechanisms by which inorganic phosphate regulates cellular protein levels, we undertook a mRNA microarray analysis of pre-osteoblast cells at 18, 21, and 24 h after inorganic phosphate exposure. Comparison of the mRNA microarray data with the 24-hour quantitative proteomic data resulted in a generally weak overall correlation; the 21-hour RNA sample showed the highest correlation to the proteomic data. However, an analysis of osteoblast relevant proteins revealed a much higher correlation at all time points. A comparison of the microarray and proteomic datasets allowed for the identification of a number of candidate proteins that are post-transcriptionally regulated by elevated inorganic phosphate, including Fra-1, a member of the activator protein-1 family of transcription factors. The analysis of the data presented here not only sheds new light on the important roles of inorganic phosphate in osteoblast function but also begins to address the contribution of post-transcriptional and post-translational regulation to a cell’s expressed proteome. The ability to accurately measure changes in both protein abundance and mRNA levels on a system-wide scale represents a novel means to extract data from previously one-dimensional datasets.

Bioactive silica-based nanoparticles stimulate bone-forming osteoblasts, suppress bone-resorbing osteoclasts, and enhance bone mineral density in vivo

Bone is a dynamic tissue that undergoes renewal throughout life in a process whereby osteoclasts resorb worn bone and osteoblasts synthesize new bone. Imbalances in bone turnover lead to bone loss and development of osteoporosis and ultimately fracture, a debilitating condition with high morbidity and mortality. Silica is a ubiquitous biocontaminant that is considered to have high biocompatibility. The authors report that silica nanoparticles (NPs) mediate potent inhibitory effects on osteoclasts and stimulatory effects on osteoblasts in vitro. The mechanism of bioactivity is a consequence of an intrinsic capacity to antagonize activation of NF-κB, a signal transduction pathway required for osteoclastic bone resorption but inhibitory to osteoblastic bone formation. We further demonstrate that silica NPs promote a significant enhancement of bone mineral density (BMD) in mice in vivo, providing a proof of principle for the potential application of silica NPs as a pharmacological agent to enhance BMD and protect against bone fracture.

New method to prepare very stable and biocompatible fluorescent silica nanoparticles

A new synthetic method has been developed to prepare fluorescent silica nanoparticles without employing isothiocyanated dye molecules and (3-aminopropyl)triethoxysilane (APS) for the thiourea linkage formation; the resulting fluorescent silica nanoparticles show excellent photochemical, thermal and pH stabilities and a good biocompatibility with over 85% viability from various cell types.

An integrated understanding of the physiological response to elevated extracellular phosphate

Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi‐phase, extracellular phosphate‐induced, signaling network in pre‐osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c‐fos. The mechanism of activation requires FGF receptor signaling followed by stimulation of N‐Ras and activation of AP‐1 and serum response elements. A distinct long‐term response also requires FGF receptor signaling and results in N‐Ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long‐term cell behavior. J. Cell. Physiol. 228: 1536–1550, 2013.

Elevated Phosphate Activates N-ras and Promotes Cell Transformation and Skin Tumorigenesis

Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously shown that elevated concentrations of phosphate increased cell proliferation and expression of protumorigenic genes such as Fra-1 and osteopontin in a preosteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage-independent transformation and proliferation of BALB/c mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and activator protein-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate and fed high- or low-phosphate diets (1.2% versus 0.2% of the diet) for 19 weeks. The high-phosphate diet increased skin papilloma number by ∼50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines reducing dietary phosphate as a novel target for chemoprevention. Cancer Prev Res; 3(3); 359–70

Identification of the Homeobox Protein Prx1 (MHox, Prrx-1) as a Regulator of Osterix Expression and Mediator of Tumor Necrosis Factor α Action in Osteoblast Differentiation

Tumor necrosis factor α (TNF‐α) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF‐treated pre‐OBs were incubated with the TNF suppressor element for protein pull‐down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14‐fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging.

LONG-TERM MONITORING OF THE PHYSICOCHEMICAL PROPERTIES OF SILICA-BASED NANOPARTICLES ON THE RATE OF ENDOCYTOSIS AND EXOCYTOSIS AND CONSEQUENCES OF CELL DIVISION

Nanomaterials are diverse in size, shape, and charge and these differences likely alter their physicochemical properties in biological systems. We have investigated how these properties alter the initial and long-term dynamics of endocytosis, cell viability, cell division, exocytosis, and interaction with a collagen extracellular matrix using silica-based fluorescent nanoparticles and the murine pre-osteoblast cell line, MC3T3-E1. Three surface modified nanoparticles were analyzed: positively charged (PTMA), negatively charged (OH), and neutrally charged polyethylene glycol (PEG). Positively charged PTMA-modified nanoparticles demonstrated the most rapid uptake, within 2 hours, while PEG modified and negatively charged OH nanoparticles demonstrated slower uptake. Cell viability was >80% irrespective of nanoparticle surface charge suggesting a general lack of toxicity. Long-term monitoring of fluorescent intensity revealed that nanoparticles were passed to daughter cells during mitotic cell division with a corresponding decrease in fluorescent intensity. These data suggest that irrespective of surface charge silica nanoparticles have the potential to internalize into pre-osteoblasts, albeit with different kinetics. Furthermore, long lived nanoparticles have the potential to be transferred to daughter cells during mitosis and can be maintained for weeks intracellularly or within a collagen matrix without toxicity and limited exocytosis.

Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)

The National Cancer Institute (NCI) has developed a Patient-Derived Models Repository (PDMR) comprised of quality-controlled, early-passage, clinically-annotated patient-derived xenografts (PDXs) to serve as a resource for public-private partnerships and academic drug discovery efforts. These models are offered to the extramural community for research use (https://pdmr.cancer.gov/), along with clinical annotation and molecular information (whole exome sequence, RNASeq), which is available in a publicly accessible database. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community9s highest priority need was preclinical models that more faithfully reflect the patient9s tumor and are associated with the patient9s treatment history. NCI has focused on generating models to complement existing PDX collections and address unmet needs in the preclinical model space. The PDMR generates the majority of its PDXs by subcutaneous implantation except for those histologies having better success rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators are posted to a public web site that houses the PDMR database. In May 2017, the public website (https://pdmr.cancer.gov/) went live with its first 100 models from histologies including pancreatic, colorectal, renal, head and neck, and lung squamous cell cancers as well as melanoma and adult soft tissue sarcomas. In early 2018, the PDMR will begin releasing models from gynecological cancers, small cell lung cancer, chondro/osteo sarcomas, lung adenocarcinoma, and squamous cell skin and Merkel cell carcinomas. In addition, wherever available germline sequence and somatic variant calls will be added to the existing molecular characterization data for each model. NCI has also increased its focus on creating PDXs from racial and ethnic minorities through several funding opportunities. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions regarding the impact of tumor heterogeneity on target qualification or clinical response, whether PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development, or if adequately powered preclinical PDX clinical trials can lead to better evaluation of therapies for future clinical use. Moving forward the PDMR plans to distribute in vitro, early-passage tumor cell cultures and cancer-associated fibroblasts as well as releasing PDX drug response data for a panel of FNA-approved therapeutic agents. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Michelle M. Gottholm Ahalt, Sergio . Y. Alcoser, Kaitlyn Arthur, Mariah Baldwin, Linda L. Blumenauer, Carrie Bonomi, Suzanne Borgel, Elizabeth Bradtke, Corinne Camalier, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Donna W. Coakley, Nicole E. Craig, Biswajit Das, Vivekananda Datta, Jordyn Davidson, Margaret R. DeFreytas, Emily Delaney, Michelle A. Eugeni, Raymond Divelbiss, Palmer Fliss, Thomas Forbes, Marion Gibson, Tara Grinnage-Pulley, Sierra Hoffman, Lilia Ileva, Paula Jacobs, Franklyn Jimenez, Joseph Kalen, Catherine Karangwa, Chris Karlovich, Candace Mallow, Chelsea McGlynn, Jenna E. Moyer, Michael Mullendore, Dianne L. Newton, Nimit Patel, Rajesh Patidar, Kevin Plater, Marianne Radzyminski, Lisa Riffle, Larry Rubinstein, Luke H. Stockwin, Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute9s patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 986.

Abstract 3789: Elevated inorganic phosphate stimulates immediate early gene expression and requires FGF receptor signaling

Dietary inorganic phosphate represents a novel chemoprevention target. Recent results from two different mouse models of tumorigenesis; the two-stage skin carcinogenesis and KrasLA1 lung tumor models suggest that a high phosphate diet increases tumor/papilloma number by approximately 50% relative to a low phosphate diet. Additionally, a low phosphate diet resulted in delayed incidence and growth of papillomas. These results suggest that increased consumption of dietary inorganic phosphate may have long-term consequences on overall health and particularly to tumorigenesis and consequently reducing dietary phosphate intake may be a novel chemoprevention strategy. Our in vitro studies have revealed an active role of inorganic phosphate in altering cell function in various cell types and suggest that the amount of available inorganic phosphate may have significant cell autonomous consequences on cell behavior including activation of ras and ERK1/2 signaling, and increased expression of transformation and metastasis associated genes such as osteopontin (spp1). However, the mechanism by which cells sense and respond to changes in extracellular phosphate resulting in increased proliferation and transformation is not fully understood. We have performed an analysis of the temporal changes in gene expression in response to elevated phosphate. Results identified the strong stimulation of immediate early genes within 15 minutes of exposure to elevated phosphate. Reporter and DNA binding assays revealed the requirement of serum response and AP-1 transcriptional elements and taken together characterize phosphate as a novel mitogen. Furthermore, we have identified the requirement of FGF receptor signaling as one of the earliest events in the phosphate signaling cascade. This finding provides a novel link between growth factor signaling, nutrient sensing and cell growth and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3789.