Faculty Bibliography

Neural crest-derived (FOb) and mesoderm-derived (POb) calvarial osteoblasts are characterized by distinct differences in their osteogenic potential. We have previously demonstrated that enhanced activation of endogenous FGF and Wnt signaling confers greater osteogenic potential to FOb. Apoptosis, a key player in bone formation, is the main focus of this study. In the current work, we have investigated the apoptotic activity of FOb and POb cells during differentiation. We found that lower apoptosis, as measured by caspase-3 activity is a major feature of neural crest-derived osteoblast which also have higher osteogenic capacity. Further investigation indicated TGF-beta signaling as main positive regulator of apoptosis in these two populations of calvarial osteoblasts, while BMP and canonical Wnt signaling negatively regulate the process. By either inducing or inhibiting these signaling pathways we could modulate apoptotic events and improve the osteogenic potential of POb. Taken together, our findings demonstrate that integration of multiple signaling pathways contribute to imparting greater osteogenic potential to FOb by decreasing apoptosis.

The mammalian skull vault, a product of a unique and tightly regulated evolutionary process, in which components of disparate embryonic origin are integrated, is an elegant model with which to study osteoblast biology. Our laboratory has demonstrated that this distinct embryonic origin of frontal and parietal bones confer differences in embryonic and postnatal osteogenic potential and skeletal regenerative capacity, with frontal neural crest derived osteoblasts benefitting from greater osteogenic potential. We outline how this model has been used to elucidate some of the molecular mechanisms which underlie these differences and place these findings into the context of our current understanding of the key, highly conserved, pathways which govern the osteoblast lineage including FGF, BMP, Wnt and TGFbeta signaling. Furthermore, we explore recent studies which have provided a tantalizing insight into way these pathways interact, with evidence accumulating for certain transcription factors, such as Runx2, acting as a nexus for cross-talk.

Biogenesis of the zona pellucida (ZP), the extracellular coat that surrounds all mammalian eggs, is a universal and essential feature of mammalian oogenesis and reproduction. The mouse egg's ZP consists of only three glycoproteins, called ZP1-3, that are synthesized, secreted, and assembled into an extracellular coat exclusively by growing oocytes during late stages of oogenesis while oocytes are arrested in meiosis. Expression of ZP genes and synthesis of ZP1-3 are gender-specific. Nascent ZP1-3 are synthesized by oocytes as precursor polypeptides that possess several elements necessary for their secretion and assembly into a matrix of long fibrils outside of growing oocytes. Failure to synthesize either ZP2 or ZP3 by homozygous null female mice precludes formation of a ZP during oocyte growth and, due to faulty folliculogenesis and a paucity of ovulated eggs, results in infertility. High-resolution structural analyses suggest that ZP glycoproteins consist largely of immunoglobulin (Ig)-like folds and that the glycoproteins probably arose by duplication of a common Ig-like domain. Mouse ZP1-3 share many features, particularly a ZP domain, with extracellular coat glycoproteins of eggs from other vertebrate and invertebrate animals whose origins date back more than 600 million years. These and other aspects of ZP biogenesis are discussed in this review.

Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of Abeta peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces beta-amyloid plaques. Our findings identify an additional pathway for the secretion of Abeta and define NEU1 as a potential therapeutic molecule for AD.

It is well known that TNFalpha play an important role in wear debris induced inflammatory osteolysis, which is a major cause of periprosthetic loosening. We previously reported that Progranulin (PGRN) antagonized TNFalpha action via binding to TNF receptors and protected against bone erosion in inflammatory arthritis (Tang W, et al, Science, 2011). The goal of this study is to determine whether PGRN plays a protective role in titanium particle induced inflammatory osteolysis. To do so, we took advantage of both in vitro RAW647.3 cells and two in vivo well-accepted mice models. Ti particle dramatically induced expression of endogenous PGRN in RAW647.3 cell and in the mice models. In addition, recombinant PGRN attenuated Ti particle induced inflammation and inflammatory osteolysis in RAW647.3 cell, calvaria explant and mice models, as the elevation of inflammatory biomarkers were significantly impaired, osteoclastogenesis was suppressed and bone erosion of calvaria was protected by PGRN. Furthermore, recombinant PGRN repressed activation of NF-B signaling pathway by Ti particle. Collectively, PGRN is a novel molecule that inhibits Ti particle induced inflammation, probably through interacting with TNF-alpha/NF-B signaling pathways. These findings not only provide novel insights into the role of PGRN in Ti particle induced inflammation reaction, but may also lead to the development of novel therapeutic intervention strategies for periprosthetic loosening

Netrins have been extensively studied for their role in axonal guidance during neural development. In addition, netrins are chemopulsants for a variety of non-neuronal cell types via binding to their receptors Unc5b and DCC. Although thought to suppress inflammation in several settings, netrin1, acting via Unc5b, inhibits macrophage migration directed by chemokines CCL2 and CCL19 to promote macrophage retention in and exacerbation of atherosclerotic plaque. We asked whether Netrin1 was expressed during osteoclast (OC) differentiation and whether it plays a role in OC differentiation.DXAscan and MicroCT analysis were performed on Netrin1 deficient mice (radiation chimeras) and wildtype (WT, radiation chimeras) littermates. OC differentiation was studied as M-CSF/RANKL-stimulated differentiation of murine bone marrow precursors to TRAP+/multinucleated cells, in the presence/absence of recombinant Netrin1 and Unc5b antibody. Netrin1, Unc5b and DCC expression were studied by RT-PCR and Western Blot in primary bone marrow-derived osteoclasts. Netrin1 immunostaining was performed in human tissue obtained following primary prosthesis implantation or after prosthesis revision. During OC differentiation cell-associated Netrin1 and Unc5b (but not DCC) protein expression increased by 30+2% and 98+4% respectively (p<0.001,n=4) and Netrin1 secretion increased by 66+2% (p<0.001,n=4). Consistently, RANKL stimulates an increase in Netrin1 and Unc5b mRNA expression during OC differentiation (25+4 and 3+0.5 fold change respectively p<0.001,n=4). Moreover, in Netrin1-deficient marrow precursors OC differentiation was diminished by 65+2% as compared to control (p<0.001,n=6), an effect reversed by addition of recombinant netrin1 to cultures (121+5% increased, p<0.5,n=4). An antibody to the netrin1 receptor Unc5b reduces OC formation by 57+6% (p<0.001, =6) whereas an antibody to DCC had no effect on OC formation (5+4% reduction, p=NS vs. control,n=6). Finally, DXAscan and MicroCT analysis demonstrated an increase in!

Progranulin (PGRN) is a multifunctional growth factor which is composed of seven-and-a-half repeats of a cysteine-rich motif in the order P-G-F-B-A-C-D-E. We previously reported that PGRN directly bound to TNF-alpha receptors (TNFR), and inhibited TNF-alpha activity (Tang, W, et al, Science. 2011). Atsttrin, an engineered protein composed of three PGRN fragments (1/2F-1/2A-1/2C), exhibited selective TNFR binding, and potently prevented inflammation in multiple arthritis mouse models (Tang, W, et al, Science. 2011). It is well established that TNF family ligands bind to receptors in a heterohexameric 3:3 complex. We created a novel engineering protein, Atsttrin primer, which comprised the same fragments as Atsttrin but in a different sequence (1/2A-1/2C-1/2F). The purpose of this study is to determine (1) whether the three fragments of Atsttrin (i.e. 1/2A, 1/2C and 1/2F) act independently for interacting with TNFR; and if so, (2) whether Atsttrin primer is able to block the binding of TNF-alpha to TNFR, and has therapeutic effect in inflammatory arthritis, as does Atsttrin. Molecular mechanistic studies through Yeast-two hybrid screening and Solid-phase binding assay revealed that Atsttrin primer had stronger binding affinity to TNFR compared with Atsttrin, and effectively inhibited TNF-alpha from binding to TNFRs. To define the role of Atsttrin primer in inflammatory arthritis, we induced collagen-induced-arthritis (CIA) model in

A common single-nucleotide polymorphism (SNP) in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This SNP is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism, we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75(NTR) and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand, which modulates neuronal morphology.