Cbf beta regulates Runx2 function isoform-dependently in postnatal bone development

Runx2 and Cbfbeta are essential for skeletal development during the embryonic stage. Runx2 has two isoforms with different N-termini. We examined the functions of the Runx2 isoforms and Cbfbeta in postnatal bone development. On luciferase and electrophoretic mobility shift assays, Runx2-I was less active than Runx2-II in the absence of Cbfb, but the two Runx2 isoforms had similar activity levels in the presence of Cbfb. We generated Runx2-I transgenic mice under the control of Col1a1 promoter and Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice. Runx2-I transgenic mice showed less severe osteopenia and fragility than Runx2-II transgenic mice due to milder inhibition of both osteoblast maturation and transition to osteocytes, even though the former mice showed higher transgene expression. However, Runx2-I/Cbfb and Runx2-II/Cbfb double transgenic mice had enhanced inhibition of osteoblast maturation, resulting in similar severity of osteopenia and fragility, although the latter mice had less osteocytes. These findings indicate that (1) Runx2-II more strongly inhibits osteoblast maturation and transition to osteocytes than Runx2-I; (2) Cbfbeta regulates Runx2 function isoform-dependently; and (3) Runx2-I activity is highly dependent on Cbfbeta. These findings demonstrate that Runx2 isoforms exert their functions through at least partly different mechanisms and Cbfbeta regulates bone development by regulating Runx2 function isoform-dependently.     

Role of Runx genes in chondrocyte differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.     

Alternate mRNA splicing in multiple human tryptase genes is predicted to regulate tetramer formation

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.     

Essential genes that regulate apoptosis

The expression of several genes has been associated with the induction of apoptosis in a wide variety of vertebrate and invertebrate organisms. However, relatively few gene products have been demonstrated to be required for cell death. This review highlights genes that are required for apoptosis and proposes mechanisms by which the proteins encoded by these genes might function.