Osteoblast intracellular localization of Nmp4 proteins

Nmp4 proteins are transcription factors that contribute to the expression of type I collagen and many of the matrix metalloproteinase genes. Numerous Nmp4 isoforms have been identified. These proteins, all derived from a single gene, have from five to eight Cys 2His 2 zinc fingers, the arrangement of which directs specific isoforms to nuclear matrix subdomains. Nmp4 isoforms also have an SH3 binding domain, typical of cytoplasmic docking proteins. Although recent evidence indicates that Nmp4 proteins also reside in the osteoblast cytoplasm, whether they localize to specific organelles or structures is not well defined. The intracellular localization of a protein is a determinant of its function and provides insights into its mechanism of action. As a first step toward determining the functional relationship between the cytoplasmic and nuclear Nmp4 compartments, we mapped their location in the osteoblast cytoplasm. Immunocytochemical analysis of osteoblasts demonstrated that Nmp4 antibodies labeled the mitochondria, colocalized with Golgi protein 58K, and lightly stained the cytoplasm. Western analysis using Nmp4 antibodies revealed a complex profile of protein bands in the nuclear, mitochondrial, and cytosolic fractions. Several of these proteins were specific to defined intracellular domains. Consistent with the western analyses, reverse transcription-polymerase chain reaction (RT-PCR) analysis detected previously uncharacterized Nmp4 isoforms. These data necessarily enlarge the known Nmp4 family from nuclear matrix transcription factors to a more widely extended class of intracellular proteins.