Faculty Bibliography

MicroRNAs (miRNAs) are a class of highly conserved small noncoding RNAs that negatively regulate gene expression by imperfectly base pairing to the 3'-untranslated region of their target mRNAs, leading to mRNA degradation or translational inhibition. The emerging field of miRNA biology has begun to unravel roles for these regulatory molecules in a variety of biological processes. This review concentrates on the roles of miRNAs in skeletogenesis as well as in skeleton-related disease processes. Before describing these data, we present a brief review of the biogenesis and action of miRNAs, the approaches to miRNAs study, and miRNAs as global regulators of development. We finish by emphasizing that the study of the biological functions of miRNAs in skeletogenesis and dysplasia represents an entirely new avenue in the exploration of bone and cartilage biology, and large gaps remain in our knowledge of miRNAs in skeletogenesis in vivo and in our knowledge of the molecular events underlying miRNA-mediated musculoskeletal disorders

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

During Drosophila oogenesis, Oskar mRNA is transported to the posterior pole of the oocyte, where it is locally translated and induces germ-plasm assembly. Oskar protein recruits all of the components necessary for the establishment of posterior embryonic structures and of the germline. Tight localization of Oskar is essential, as its ectopic expression causes severe patterning defects. Here, we show that the Drosophila homolog of mammalian Lasp1 protein, an actin-binding protein previously implicated in cell migration in vertebrate cell culture, contributes to the accumulation of Oskar protein at the posterior pole of the embryo. The reduced number of primordial germ cells in embryos derived from lasp mutant females can be rescued only with a form of Lasp that is capable of interacting with Oskar, revealing the physiological importance of the Lasp-Oskar interaction.

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCalpha and PKCepsilon isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-beta (Abeta) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCalpha down-regulation; (ii) PKCepsilon up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Abeta. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Abeta generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Abeta generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity

The success of lower extremity microsurgical reconstructions may be compromised postoperatively secondary to several factors, including thrombosis, infection, bleeding, and edema. To address edema, surgeons may use protocols for gradually dangling and/or wrapping the affected extremity. Such protocols vary widely among surgeons and are typically based on training and/or prior experience. To that end, we distributed surveys to five plastic surgeons who are experienced in microvascular lower extremity reconstruction at five different institutions. The surveys inquired about postoperative management protocols for lower extremity free flaps with regard to positioning, compression, initiation and progression of postoperative mobilization, nonweightbearing and weightbearing ambulation, assessment of flap viability, and flap success rate. These protocols were then evaluated for similarities to create a consensus of postoperative management guidelines. Progressive periods of leg dependency and compression therapy emerged as important elements. Although the consensus protocol developed in this study is considered safe by each participant, we do not intend for these recommendations to serve as a standard of care, nor do we suggest that any one particular protocol leads to improved outcomes. However, these recommendations may serve as a guide for less experienced surgeons or those without a protocol in place

Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, though the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cellderived factor-1 (SDF-1), a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs, but these cells did assume an endothelial phenotype as evidenced by their ability to tubularize in Matrigel; the presence of endothelial cells markedly increased ASC tubularization. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis. __________________________________________________________________________ ____ Author contributions: H.T.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; I.N.V.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; E.C.: Collection and assembly of data, data analysis and interpretation; S.El-ftesi: Collection and assembly of data, data analysis and interpretation; M.J.: Data analysis and interpretation, manuscript writing; E.I.C.: Collection and assembly of data, data analysis and interpretation; J.P.: Collection and assembly of data, data analysis and interpretation; E.N.: Collection and assembly of data, data analysis and interpretation; M.T.L.: Conception and design, data analysis and interpretation, final approval of manuscript; G.C.G.: Conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. Hariharan Thangarajah and Ivan N. Vial contributed equally to this work