Faculty Bibliography

The principal function of an epithelium is to form a dynamic barrier that regulates movement between body compartments. Each epithelium is specialized with barrier functions that are specific for the tissues it serves. The apical surface commonly faces a lumen, but the retinal pigment epithelium (RPE) appears to be unique by a facing solid tissue, the sensory retina. Nonetheless, there exists a thin (subretinal) space that can become fluid filled during pathology. RPE separates the subretinal space from the blood supply of the outer retina, thereby forming the outer blood-retinal barrier. The intricate interaction between the RPE and sensory retina presents challenges for learning how accurately culture models reflect native behavior. The challenge is heightened by findings that detail the variation of RPE barrier proteins both among species and at different stages of the life cycle. Among the striking differences is the expression of claudin family members. Claudins are the tight junction proteins that regulate ion diffusion across the spaces that lie between the cells of a monolayer. Claudin expression by RPE varies with species and life-stage, which implies functional differences among commonly used animal models. Investigators have turned to transcriptomics to supplement functional studies when comparing native and cultured tissue. The most detailed studies of the outer blood-retinal barrier have focused on human RPE with transcriptome and functional studies reported for human fetal, adult, and stem-cell derived RPE.

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signalling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signalling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood-brain barrier. Local insulin/IGF signalling from glial cells, the blood-brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signalling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

BACKGROUND:: Scarring represents a significant biomedical burden in clinical medicine. Mechanomodulation has been linked to scarring through inflammation, but until now a systematic approach to attenuate mechanical force and reduce scarring has not been possible. METHODS:: We conducted a twelve month, prospective, open label, randomized, multi-center clinical trial to evaluate abdominoplasty scar appearance following post-operative treatment with the embrace device to reduce mechanical forces on healing surgical incisions. Incisions from 65 healthy adult subjects were randomized to receive embrace treatment on one half of an abdominoplasty incision and control treatment (surgeon's optimal care methods) on the other half. The primary endpoint for this study was the difference between assessments of the scar appearance for the treated and control sides using the Visual Analogue Scale (VAS) scar score. RESULTS:: Final twelve month study photos were obtained from 36 subjects who completed at least five weeks of dressing application. The mean VAS score for embrace-treated scars (2.90) was significantly improved compared to control-treated scars (3.29) at 12 months (difference = 0.39, 95% confidence interval [0.14, 0.66], p = 0.027). Both subjects and investigators found that embrace-treated scars demonstrated significant improvements in overall appearance at 12 months using the Patient and Observer Scar Assessment Scale (POSAS) evaluation (p = 0.02 and p < 0.001, respectively). No serious adverse events were reported. CONCLUSION:: These results demonstrate that the embrace device significantly reduces scarring following abdominoplasty surgery. To our knowledge, this represents the first level one evidence for post-operative scar reduction.

Canonical Hedgehog (HH) signaling leads to the regulation of the GLI code: the sum of all positive and negative functions of all GLI proteins. In humans, the three GLI factors encode context-dependent activities with GLI1 being mostly an activator and GLI3 often a repressor. Modulation of GLI activity occurs at multiple levels, including by co-factors and by direct modification of GLI structure. Surprisingly, the GLI proteins, and thus the GLI code, is also regulated by multiple inputs beyond HH signaling. In normal development and homeostasis these include a multitude of signaling pathways that regulate proto-oncogenes, which boost positive GLI function, as well as tumor suppressors, which restrict positive GLI activity. In cancer, the acquisition of oncogenic mutations and the loss of tumor suppressors - the oncogenic load - regulates the GLI code toward progressively more activating states. The fine and reversible balance of GLI activating GLIA and GLI repressing GLIR states is lost in cancer. Here, the acquisition of GLIA levels above a given threshold is predicted to lead to advanced malignant stages. In this review we highlight the concepts of the GLI code, the oncogenic load, the context-dependency of GLI action, and different modes of signaling integration such as that of HH and EGF. Targeting the GLI code directly or indirectly promises therapeutic benefits beyond the direct blockade of individual pathways.

Differences in epidermal melanin levels result in the wide variation in color associated with ethnic skin diversity. Ethnic differences result from variance in melanogenesis, melanin transfer, and melanosome degradation in the epidermis, but the mechanisms underlying these differences, and their contribution to the regulation of skin color, are not fully understood. In this study, we explored proteins which were differentially expressed between Caucasian- and African American donor skin- derived melanocytes using twodimensional gel electrophoresis and mass spectroscopy. We consequently identified an important role for Heat Shock Protein 70 (Hsp70 encoded by HSPA1A) in skin color determination. In contrast to a previous report suggesting negative regulation of melanogenesis, Hsp70 was found to be more abundant in melanocytes from African American donors compared to those from Caucasian donors, which also reflected protein expression in the skin. In particular, inhibition of Hsp70, using either a specific inhibitor or siRNA targeting, significantly decreased melanin content of African American donor melanocytes, consistent with previous reports that Hsp70 is an Endoplasmic Reticulum (ER) chaperone involved in post-translational modification of tyrosinase. Indeed, inhibition of Hsp70 caused a decrease in tyrosinase protein levels by affecting its maturation. Taken together, our data reveal that Hsp70 plays an essential role in regulation of melanogenesis and thereby contributes to the ethnic diversity of skin color

Bone morphogenetic protein 2(BMP2) is known to activate unfolded protein response (UPR) signal molecules in chondrogenesis. Inositol-requiring enzyme-1alpha (IRE1alpha),as one of three unfolded protein sensors in UPR signaling pathways, can be activated during ER stress. However, the influence on IRE1alpha in chondrocyte differentiation has not yet been elucidated. Here we present evidence demonstrating that overexpression of IRE1alpha inhibits chondrocyte differentiation, as revealed by reduced expression of collagen II (ColII), Sox9, collagen X (ColX), matrix metalloproteinase 13 (MMP-13), Indian hedgehog (IHH), Runx2 and enhanced expression of parathyroid hormone-related peptide (PTHrP). Furthermore, IRE1alpha-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. The RNase and Kinase domains of IRE1alpha C-terminal are necessary for its full enzymatic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that granulin-epithelin precursor(GEP), a growth factor known to stimulate chondrogenesis, induced IRE1alpha expression in chondrogenesis. The expression of IRE1alpha is depended on GEP signaling, and IRE1alpha expression is hardly detectable in GEP-/- embryos. In addition, IRE1alpha inhibits GEP-mediated chondrocyte differentiation as a negative regulator. Altered expression of IRE1alpha in chondrocyte hypertrophy was accompanied by altered levels of IHH and PTHrP. Collectively, IRE1alpha may be a novel regulator of chondrocyte differentiation by 1) inhibition GEP-mediated chondrocyte differentiation as a negative regulator; 2) promoting IHH/PTHrP signaling.

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.

The unfolded protein response (UPR) - the endoplasmic reticulum stress response - is found in various pathologies including ischemia-reperfusion injury (IRI). However, its role during IRI is still unclear. Here, by combining two different bioinformatical methods - a method based on ordinary differential equations (Time Series Network Inference) and an algebraic method (probabilistic polynomial dynamical systems) - we identified the IRE1alpha-XBP1 and the ATF6 pathways as the main UPR effectors involved in cell's adaptation to IRI. We validated these findings experimentally by assessing the impact of their knock-out and knock-down on cell survival during IRI.