Divergent activities of osteogenic BMP2, and tenogenic BMP12 and BMP13 independent of receptor binding affinities.

Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.

Proteomic identification and functional validation of activins and bone morphogenetic protein 11 as candidate novel muscle mass regulators.

Myostatin is a secreted TGF-beta family member that controls skeletal muscle growth. Humans, cattle, and dogs carrying natural loss-of-function mutations in the myostatin gene and myostatin knockout mice exhibit significant increases in skeletal muscle mass. Treatment of adult mice with antimyostatin antibodies also resulted in significant muscle mass increases. However, myostatin-knockout mice that were treated with a soluble form of the activin type II receptor (ActRII) B increased their muscle mass by an additional 15-25%, indicating that there is at least one additional ligand, in addition to myostatin, that functions to limit muscle growth. Here, both soluble ActRII and -IIB fragment-crystallizable proteins were used to affinity purify their native ligands from human and mouse sera. Using mass spectrometry-based proteomics and in vitro binding assays we have identified and confirmed that a number of TGF-beta family members, including myostatin, activins-A, -B, and -AB, bone morphogenetic proteins (BMPs) -9, -10, and -11, bind to both ActRIIs. Many of these factors, such as BMPs-11, -9, and -10 were discovered in systemic circulation for the first time, indicating that these ligands may also act in an endocrine fashion. Using a promoter-specific gene reporter assay, we demonstrated that soluble ActRIIB fragment-crystallizable proteins can inhibit the canonical signaling induced by these ligands. In addition, like myostatin, these factors were able to block the differentiation of myoblast cells into myotubes. However, in addition to myostatin, only BMP-11, and activins-A, -B, and -AB could be blocked from inhibiting the myoblast-to-myotube differentiation with both soluble ActRIIs, thus implicating them as potential novel regulators of muscle growth.

Influence of EBV on the peripheral blood memory B cell compartment.

Peripheral blood memory B cells latently infected with EBV bear somatic mutations and are typically isotype switched consistent with being classical Ag-selected memory B cells. In this work, we performed a comparative analysis of the expressed Ig genes between large sets of EBV-infected and uninfected peripheral blood B cells, isolated from the same infectious mononucleosis patients, to determine whether differences exist that could reveal the influence of EBV on the production and maintenance of these cells. We observed that EBV(+) cells on average accumulated more somatic hypermutations than EBV(-) cells. In addition, they had more replacement mutations and a higher replacement-silent ratio of mutations in their CDRs. We also found that EBV occupies a skewed niche within the memory compartment, due to its exclusion from the CD27(+)IgD(+)IgM(+) subset, but this skewing does not affect the overall structure of the compartment. These results indicate that EBV impacts the mutation and selection process of infected cells but that once they enter memory they cannot be distinguished from uninfected cells by host homeostasis mechanisms.

Peripheral B cells latently infected with Epstein-Barr virus display molecular hallmarks of classical antigen-selected memory B cells.

Epstein-Barr virus (EBV) establishes a lifelong persistent infection within peripheral blood B cells with the surface phenotype of memory cells. To date there is no proof that these cells have the genotype of true germinal-center-derived memory B cells. It is critical to understand the relative contribution of viral mimicry versus antigen signaling to the production of these cells because EBV encodes proteins that can affect the surface phenotype of infected cells and provide both T cell help and B cell receptor signals in the absence of cognate antigen. To address these questions we have developed a technique to identify single EBV-infected cells in the peripheral blood and examine their expressed Ig genes. The genes were all isotype-switched and somatically mutated. Furthermore, the mutations do not cause stop codons and display the pattern expected for antigen-selected memory cells based on their frequency, type, and location within the Ig gene. We conclude that latently infected peripheral blood B cells display the molecular hallmarks of classical antigen-selected memory B cells. Therefore, EBV does not disrupt the normal processing of latently infected cells into memory, and deviations from normal B cell biology are not tolerated in the infected cells. This article provides definitive evidence that EBV in the peripheral blood persists in true memory B cells.