Faculty Bibliography

TGFbeta is secreted in a latent state and must be "activated" by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFbeta mobilisation in vivo. Here we review the process of TGFbeta secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFbeta is secreted, sequestered and released. This allows the separation of more ancient TGFbeta activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

Pigmentation disorders span the genetic spectrum from single-gene autosomal recessive disorders such as oculocutaneous albinism (OCA), the autosomal dominant disorder piebaldism to X-linked ocular albinism and multifactorial vitiligo. OCA connotes a group of disorders that result in hypopigmented skin due to decreased melanin production in melanocytes and loss of visual acuity. There are four non-syndromic forms, OCA1-4, which are classified based on the gene that is mutated (tyrosinase, OCA2, tyrosinase-related protein 1 and SLC45A2, respectively). Despite the fact that multiple genes account for the various forms of OCA, the phenotypes of all four forms result from disruption in the maturation and trafficking of the enzyme tyrosinase. OCA2 is the most prevalent autosomal recessive disorder among southern African blacks, affecting 1/3 900 individuals; while OCA3, although rare, is most prevalent in southern Africa. Another common pigmentation disorder in southern Africa is vitiligo, which affects 1 - 2% of people worldwide. Vitiligo is a complex, acquired disorder in which melanocytes are destroyed due to an autoimmune response. The aetiology underlying this disorder is poorly understood, although recent genetic association studies have begun to shed light on the contributing factors. Pigmentation disorders have significant psychosocial implications and co-morbidities, yet therapies are still lacking. Recent progress in our understanding of the pathobiology of both albinism and vitiligo may herald novel treatment strategies for these disorders.

The basal (ligand-independent) kinase activity of receptor tyrosine kinases (RTKs) promotes trans-phosphorylation on activation loop tyrosines upon ligand-induced receptor dimerization, thus upregulating intrinsic kinase activity and triggering intracellular signaling. To understand the molecular determinants of intrinsic kinase activity, we used X-ray crystallography and NMR spectroscopy to analyze pathogenic FGF receptor mutants with gradations in gain-of-function activity. These structural analyses revealed a "two-state" dynamic equilibrium model whereby the kinase toggles between an "inhibited," structurally rigid ground state and a more dynamic and heterogeneous active state. The pathogenic mutations have different abilities to shift this equilibrium toward the active state. The increase in the fractional population of FGF receptors in the active state correlates with the degree of gain-of-function activity and clinical severity. Our data demonstrate that the fractional population of RTKs in the active state determines intrinsic kinase activity and underscore how a slight increase in the active population of kinases can have grave consequences for human health.

Endothelial dysfunction is associated with diverse cardiovascular pathologies. Here, we show a previously unappreciated role for the Abelson (Abl) family kinases (Abl and Arg) in endothelial function and the regulation of angiogenic factor pathways important for vascular homeostasis. Endothelial Abl deletion in Arg-null mice led to late-stage embryonic and perinatal lethality, with mutant mice displaying focal loss of vasculature and tissue necrosis. Loss of Abl kinases led to increased endothelial cell apoptosis both in vitro and in vivo, contributing to vascular dysfunction, infarction, and tissue damage. Mechanistically, we identify a unique dual role for Abl kinases in the regulation of angiopoietin/Tie2 protein kinase signaling. Endothelial Abl kinases modulate Tie2 expression and angiopoietin-1-mediated endothelial cell survival. These findings reveal a critical requirement for the Abl kinases in vascular development and function, which may have important implications for the clinical use of Abl kinase inhibitors.

Here, we show that apolipoprotein A1 (apoA1), the major protein component of high density lipoprotein (HDL), through both innate and adaptive immune processes, potently suppresses tumor growth and metastasis in multiple animal tumor models, including the aggressive B16F10L murine malignant melanoma model. Mice expressing the human apoA1 transgene (A1Tg) exhibited increased infiltration of CD11b(+) F4/80(+) macrophages with M1, anti-tumor phenotype, reduced tumor burden and metastasis, and enhanced survival. In contrast, apoA1-deficient (A1KO) mice showed markedly heightened tumor growth and reduced survival. Injection of human apoA1 into A1KO mice inoculated with tumor cells remarkably reduced both tumor growth and metastasis, enhanced survival, and promoted regression of both tumor and metastasis burden when administered following palpable tumor formation and metastasis development. Studies with apolipoprotein A2 revealed the anti-cancer therapeutic effect was specific to apoA1. In vitro studies ruled out substantial direct suppressive effects by apoA1 or HDL on tumor cells. Animal models defective in different aspects of immunity revealed both innate and adaptive arms of immunity contribute to complete apoA1 anti-tumor activity. This study reveals a potent immunomodulatory role for apoA1 in the tumor microenvironment, altering tumor-associated macrophages from a pro-tumor M2 to an anti-tumor M1 phenotype. Use of apoA1 to redirect in vivo elicited tumor-infiltrating macrophages toward tumor rejection may hold benefit as a potential cancer therapeutic.

BACKGROUND: Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity. METHODS: Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]). RESULTS: Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls. CONCLUSIONS: Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals. CLINICAL RELEVANCE: We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Delta/Delta) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of gammaH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed "TaDa," a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates.

Connexins form a family of transmembrane proteins that consists of 20 members in humans and 21 members in mice. Six connexins assemble into a connexon that can function as a hemichannel or connexon that can dock to a connexon expressed by a neighbouring cell, thereby forming a gap junction channel. Such intercellular channels synchronize responses in multicellular organisms through direct exchange of ions, small metabolites, and other second messenger molecules between the cytoplasms of adjacent cells. Multiple connexins are expressed in the cardiovascular system. These connexins not only experience the different biomechanical forces within this system, but may also act as effector proteins in co-ordinating responses within groups of cells towards these forces. This review discusses recent insights regarding regulation of cardiovascular connexins by mechanical forces and junctions. It specifically addresses effects of (i) shear stress on endothelial connexins, (ii) hypertension on vascular connexins, and (iii) changes in afterload and the composition of myocardial mechanical junctions on cardiac connexins.