Carolina M. Montaño Intracellular localization of a novel BMP-2 variant protein involved in Col11a2 regulation Faculty Mentor: Laura C. Bridgewater, Microbiology and Molecular Biology Cartilage is a type of connective tissue that is found in many parts of the skeleton and covers the joint surfaces of bones. It consists of an abundant extracellular matrix (ECM) that is produced and maintained by chondrocytes. This ECM includes type II, IX and XI collagens. Collagen XI plays a critical role in regulating fibril diameter and cartilage development. This heterotrimeric molecule consists of three chains—α1(XI), α2(XI), and α3(XI)—encoded by three distinct genes, Col11a1, Col11a2, and Col2a1, respectively.1 While expression of the α1(XI) and α3(XI) subunits is found in other tissues, the expression of the α2(XI) is restricted to chondrocytes. Dr. Bridgewater’s laboratory uses the Col11a2 gene as a model system to study chondrocyte- specific gene regulation. This laboratory has identified three chondrocyte-specific enhancer elements capable of directing gene expression.1,2 Her work focuses on the identification of novel transcription factors that bind to the enhancers and thus regulate the expression of the collagen gene. Recently, a novel variant of the bone morphogenetic protein-2 (BMP-2) was identified in her lab as a potential transcriptional regulator of the Col11a2 gene. The purpose of my project was to elucidate the subcellular localization of this novel peptide using immunofluorescence and confocal laser microscopy. Bone morphogenetic proteins (BMPs) are growth factors that have been shown to play an important role in embryonic development and particularly in cartilage and bone formation. So far research has shown that these proteins bind to an extracellular receptor and activate signal transduction pathways that ultimately lead to nuclear translocation of other DNA-binding proteins to regulate gene transcription. However, preliminary data obtained in Dr. Bridgewater’s lab suggests that a BMP-2 variant protein may itself be translocated to the nucleus, where it binds directly to DNA to regulate transcription. The cellular processing of BMPs includes their translation in prepropeptide form, glycosylation, and cleavage to produce a mature C-terminal region and an N-terminal propeptide. The C- terminal mature segment is secreted as a dimer that can have autocrine and paracrine functions during osteoblast differentiation.3 The propeptide is thought to be degraded, but it has two potential nuclear localization signals (NLS) in its sequence. NLSs interact with other cytoplasmic proteins to shuttle proteins into the nucleus. One of the NLS is located in the N- terminal of the propeptide whereas the other one, containing a bipartite motif, is located where the prepropeptide is cleaved to release the N-terminal propeptide and the C-terminal mature region. To determine whether the novel BMP-2 variant protein can be detected in the nucleus of chondrocytes, I transfected cells with expression plasmids containing the complete BMP-2 coding sequence, only the propeptide region, and only the mature region. I then incubated chondrocytes with primary antibodies generated against the mature region and two epitopes from the propeptide region of BMP-2. I used TRITC-labeled secondary antibodies to visualize where the primary antibody bound using confocal microscopy. I also ran experiments using primary antibodies against BMP receptors because they also have two NLSs on them. Unfortunately, the nucleus as well as the cytoplasm fluoresced using this approach, making it impossible for me to asses whether the propeptide was shuttled to the nucleus or not. To solve this problem, we inserted an epitope to tag the BMP proteins we wanted to express. This epitope, a myc tag, was recognized by a primary myc antibody and visualized using the secondary antibody we used previously. Even though this approach usually reduces background staining significantly, in our case it produced staining both in the cytoplasm and in the nucleus. We had decided to use antibodies instead of a green-fluorescent protein (GFP) tag on the BMP-2 variant because GFP is large and might interfere with BMP’s multi-step processing. Because the other approaches did not work, we decided to make constructs that had both NLSs present in the propeptide region attached to the sequence of GFP. If the propeptide region uses these NLSs to be shuttled to the nucleus, it can be localized directly and no antibody staining is needed. So far we have discovered that the bipartite NLS is sufficient to transport GFP to the nucleus, and that a mutation in this NLS blocks this transport. Further experiments will show the effect of the N- terminal signal and what occurs to BMP when the cleavage of the propeptide is prevented. These experiments will also have the GFP tag to be visualized. Ultimately, if we can confirm that BMP-2 is transported to the nucleus, our group will design constructs that have the NLS mutated so that the localization in the nucleus can be blocked without disrupting secretion of the extracellular form of the protein. This construct would then be modified and used to create a mouse model in which nuclear BMP-2 is blocked. The demonstration of a variant BMP-2 protein in the nucleus could foster the investigation of other BMPs and justify taking a closer look at the processing and localization of other propeptides that are present in chondrocytes. Therefore, this work can not only provide new knowledge in the field but also has the potential to trigger a drastic reexamination of the role played by all BMPs. Unfortunately, I will not be able to be part of it anymore because I graduated in April, but I am very excited to see the results of such an exciting project. BIBLIOGRAPHY 1. Bridgewater, L.C., Lefebvre, V., and de Crombrugghe, B. (1998). Chondrocyte- specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specific enhancer. The Journal of Biological Chemistry, 273, 14998-15006. 2. Bridgewater, L.C., Walker, M.D., Miller, G.C., Ellison, T.A., Holsinger, L.D., Potter, J.L., Jackson, T.L., Chen, R.K., Winkel, V.L., Zhang, Z., McKinney, S., de Crombrugghe, B.(2003). Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements. Nucleic Acids Research, 31, 1541 – 1553. 3. Chen, D., Zhao, M., Harris, S., and Mi, Z. (2004). Signal transduction and biological functions of bone morphogenetic proteins. Frontiers in Bioscience, 9, 349-358.
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