My research program utilizes cutting-edge proteomic technologies as well as molecular biological approaches to address the following biological questions and thematics as they pertain to bone research:

1. Biomarker Discovery and Drug Biodistribution

Protein biomarkers that predict the course of disease and the therapeutic response are essential for early disease diagnosis and for the development of patient-specific therapies. My research group employs MALDI MS to directly analyze protein profiles from human tissue specimens or animal models to discover disease-specific biomolecules that will augment disease diagnosis, prognosis and provide novel therapeutic targets for disease intervention. Two main modes of in situ proteomic assessment are conducted routinely in my group: (1) MALDI tissue profiling and (2) molecular imaging by MALDI MS (IMS).

Tissue profiling MALDI MS

This technology can be performed on intact tissue sections for the determination of the spatial distribution of compounds and their relative expression levels without the need for molecularly specific exogenous compounds such as antibody-based reagents. Signals can be mapped to discrete tissue regions, thereby adding a new dimension to protein analysis. Ongoing collaborative projects with our clinical colleagues utilize tissue profiling to identify and characterize disease-specific proteins for osteoporosis, osteoarthritis, bone cancer metastasis and wound healing. As demonstrated in Figure 1, sample preparation for tissue profiling MALDI MS begins with tissue sectioning, depositing droplets of matrix to specific regions of the tissue specimen, insertion of the specimen into a MALDI time-of-flight mass spectrometer, and ablation of proteins crystallized in the matrix with a nitrogen laser. Each protein detected by the mass spectrometer is registered as one specific protein peak, generating a “protein profile” for that region of the tissue.

Figure 1

Figure 3 (A) Optical image of the section before matrix application. The area that contains the tumor is outlined in red. (B-L) Ion density maps obtained at different m/z values with an imaging resolution of 110 µm. The ion density maps are depicted as pseudocolor images with white representing the highest protein concentration and black the lowest.

Molecular Imaging by MALDI MS (IMS).

As demonstrated in Figure 1, preparation for IMS is very similar to tissue profiling, except that matrix is either spray-coated or robotically deposited (~150 µm resolution) across the entire tissue section. Imaging the tissue section involves generation of an ordered array of spots or pixels so that multiple molecular images can be produced from a tissue section. We differentiate this technique from tissue profiling, where only several selected regions on a tissue are of interest. For IMS, the entire tissue section is under a fixed laser beam over a predetermined two-dimensional array or grid, generating a full mass spectrum at each grid coordinate. Software has been optimized to automate the scanning process, including fast data acquisition, online compression, and image reconstruction. 2D intensity maps is then reconstructed to provide specific molecular images of a tissue. Typically, 20–100 laser shots are averaged to provide a spectrum at every image coordinate pixel. The mass spectrum displays well over 500 distinct signals in a molecular mass range up to 100,000 Da. Protein ion density maps (or images) are obtained by displaying the intensities of specific mass-to-change values in two-dimensional space. Depending on the requirements of the analysis, image resolution can be chosen by changing the distance between the pixels. IMS has distinct advantages over traditional techniques in ascertaining protein localization because of its unique molecular specificity. We currently employ IMS to understand the biodistribution of proteins and small molecular therapeutics for several bone diseases, including bone cancer and osteoporosis. Figure 3 shows an IMS analysis of a mouse brain with a tumor. Localization and intensity of hundreds of proteins were detected, a few of which are shown here. IMS allows localization and relative abundance of small molecular therapeutics as well as hundreds of protein in intact tissue specimens.

2. The Transcriptional Regulation of Bone Morphogenetic Protein Receptors (BMPR)

Figure 5

This arm of my program focuses on the transcriptional regulation of BMP receptors using both molecular biological and proteomic tools. BMPRs are members of the transforming growth factor-beta receptor family of receptors and are expressed on the surface of several cell types including endothelial cells, macrophages and osteoblasts. Recently, a cause for familial primary pulmonary hypertension (FPPH) has been identified as mutations in the gene encoding BMPR2, and we have shown that HIV-1 represses BMPR2 transcription and diminishes SMAD signaling in macrophages. We have also shown that BMPR2 transcription from the basal promoter is dependent on cyclin-dependent kinase 9 and cyclin T. Several groups have demonstrated altered BMP signaling in various forms of cancer such as breast and prostate. To this end, we are interested in BMP signaling in cancer and intracellular signaling pathways that drive BMP signaling. Promoter analyses (Figure 5) reveal several transcription factor binding sites that could modulate BMPR2 expression in cancer and other bone metabolic diseases. These elements are currently under investigation by our group.

Research Techniques:

Proteomic technologies are routinely utilized in our projects.  They include tissue profiling matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), protein imaging by MALDI MS (IMS), 2-dimensional difference gel electrophoresis (2D-DIGE), and liquid chromatography MS (LC-MS).  These techniques are employed for global or specific assessment of protein expression patterns, detection of biomarkers, and characterization of protein and drug biodistribution in intact tissues.

Molecular biological techniques are also employed in our research.  They include western and northern blotting, PCR, cell culture, transfection, immunohistochemistry, DNA cloning, flow cytometry, and microscopy.

Recent Publications:

Caldwell, R.L., Opalenik, S.R., Davidson, J.M., Caprioli, R.M., Nanney, L.B. 2006. Tissue profiling by MALDI mass spectrometry reveals prominent calcium-binding proteins in the proteome of regenerative MRL mouse wounds.  Journal of Investigative Dermatology.  Submitted.

Holt, G.E., Schwartz, H.S., Caldwell, R.L.  2006. Tissue profiling in musculoskeletal oncology by MALDI mass spectrometry.  Clinical Orthopaedics and Related Research.  In Press.

Nanney, L.B., Caldwell, R.L., Pollins, A.C., Cardwell, N.L., Opalenik, S., Davidson, J.M.   2006. Novel approaches for understanding the mechanism of wound repair. Journal of Investigative Dermatology. In Press.

Caldwell, R.L., Gonzalez, A., Olson, S.J., Oppenheimer, S.R., and Caprioli, R.M.  2006.  Molecular determination of tumor margins by protein imaging mass spectrometry.  Cancer Cell (submitted).

Caldwell, R.L., Holt, G.E. and Caprioli, R.M. 2005.  Protein profiling by MALDI mass spectrometry distinguishes clinical grades of soft tissue sarcomas.  Cancer Genomics and Proteomics.  2(6):333-346.

Caldwell, R.L. and Caprioli, R.M.  2005. Tissue profiling by mass spectrometry:  A review of methodology and applications.  Molecular and Cellular Proteomics. 4(4):394-401.

Caldwell, R.L., Lane, K.D., Gaddipati, R. and Shepherd, V.L. 2006. HIV-1 Tat represses transcription of the bone morphogenetic protein receptor-2 in U937 monocytic cells.  The Journal of Leukocyte Biology. 79(1):192-201.

Caldwell, R.L., Lane, K.D., and Shepherd, V.L.  2005 HIV-1 Tat and cyclin T interaction represses transcription of the mannose receptor and the bone morphogenetic protein receptor-2 in macrophages.  Archives of Biochemistry and Biophysics.  In Press.

Reyzer, M. L., Caldwell R. L., Dugger T.C., Forbes J.T., Ritter C.A., Guix M., Arteaga C.L., Caprioli R.M. 2004. Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics. Cancer Research. 64(24):9093-100.

Caldwell, R.L., Egan, B.S., and Shepherd, V.L.   2000.  HIV-1 Tat represses transcription from the mannose receptor promoter.  The Journal of Immunology. 165(12):7035-41

Book Chapters:

Caldwell, R.L. and Caprioli, R.M. 2004. The Encyclopedia of Mass Spectrometry.  Vol. 2:  Biological Applications.  Chapter:  Protein Markers of Health and Disease.  Elsevier Publishing Company. 

Caldwell, R.L. and Caprioli, R.M. 2005. Proteomics in Cancer Research:  Concepts and Methods for Basic and Clinical Applications. Chapter: Tissue Profiling and Imaging by Matrix Assisted Laser Desorption Ionization Mass Spectrometry. John Wiley & Sons.

Societies and Memberships

2001-Current:  American Society for Biochemistry and Molecular Biology
http://asbmb.org/

2005-Current:  American Association for Cancer Research
http://www.aacr.org/

2006-Current:  American Society of Bone and Mineral Research 
http://asbmr.org/

2006-Current:  Orthopaedic Research Society
http://www.ors.org/

University Affiliations

Vanderbilt-Ingram Comprehensive Cancer Center, Associate Member
http://www.mc.vanderbilt.edu/vicc/

Vanderbilt Graduate Faculty
http://bret.mc.vanderbilt.edu/bret/

Vanderbilt Skin Diseases Center
http://www.mc.vanderbilt.edu/centers/sdrcc/

Vanderbilt Center for Matrix Biology
http://www.mc.vanderbilt.edu/cmb/

Mass Spectrometry Research Center: 
http://www.mc.vanderbilt.edu/msrc/

Vanderbilt Center for Bone Research
http://bonecenter.mc.vanderbilt.edu

Invited Oral Presentations

Caldwell, R.L.  October 2005.  Proteomic evaluation of sarcoma and bone metabolic diseases.  Department of Orthopaedics and Sports Medicine, University of Washington, Seattle.

Caldwell, R.L.  June 2005.  Molecular assessment of tumor margins by imaging mass spectrometry.  American Society of Mass Spectrometry.  San Antonio, TX.

Caldwell, R.L.  May 2005.  Proteomic evaluation of soft tissue sarcomas: Biomarker discovery and tumor margin assessment.  Musculoskeletal Tumor Society. Nashville, TN.

Caldwell, R.L.  April 2005.  Applications of tissue profiling and imaging mass spectrometry: Proteomic tools for biomedical research. Bristol-Myers Squibb Pharmaceutical Research Institute; Princeton, NJ.

Caldwell, R.L.  March 2005.  Tissue profiling and imaging mass spectrometry:  proteomic tools for cancer research.  The Burnham Institute; La Jolla, CA. 

Caldwell, R.L., Lane, K.B., and Shepherd, V.L. April 2002.  HIV-1 Tat and Vpr synergistically repress the mannose receptor and the bone morphogenetic protein receptor-2 transcription in macrophages by interaction with cyclin T.  FASEB International Conference, Experimental Biology:  American Society of Biochemistry and Molecular Biology; New Orleans, LA.