Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

ROLE OF CAVEOLIN-3 IN THE MODULATION OF HERG POTASSIUM CHANNEL

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr) that is important for cardiac repolarization. Previously, we have discovered that hERG channels rapidly internalize in low extracellular K+ ([K+]o). In cell culture, this process is driven by the endocytic protein, caveolin-1 (Cav1), which is an integral player in the caveolae-dependant endocytosis pathway. However, in the heart, Caveolin-3 (Cav3) is, in fact, the predominant form in the myocyte, and thus may play a direct role in regulating hERG expression in the heart. Thus, I hypothesize that this reduction of hERG conductance in cardiac myocytes derives from the presence of Cav3, which is integral regulator of hERG homeostasis innately in the heart. To investigate the effect of Cav3 on hERG, I overexpressed Cav3 in human embryonic kidney 293 (HEK-293) cells stably expressing hERG channels. Cav3 overexpression significantly and specifically decreased both the hERG current amplitude and the mature channel expression in normal culture conditions. Co-immunoprecipitation analysis and confocal imaging demonstrated an association between hERG and Cav3 in HEK cells as well as rat and rabbit cardiomyocytes. Mechanistically, I discovered that Cav3 possesses a faster turnover rate compared to Cav1, and can enhance hERG degradation through up-regulating mature channel ubiquitination via the ubiquitin ligase, NEDD4-2. Knockdown of Cav3 in neonatal cardiac myocytes also enhanced hERG expression. My data indicate that Cav3 participates in hERG trafficking, and is an important regulator of hERG channel homeostasis in cardiac myocytes. This information provides a platform for future intervention of the hERG-induced type-2 long QT syndrome (LQTS).

"The Black Imprint of Sandals in White Mosaic Floors": H.D.'s Mythomystical Poetics

My dissertation examines the traces of inverse (mytho)mysticism, more synchronous with mythical alchemy than transcendent mystery, in H.D.’s mature work (1946-1961). Whereas H.D.’s earliest works respond to a fin de siècle occultism and a collective psyche troubled by the eschatological distress that, as Susan Acheson writes, “was widespread amongst modernist writers grappling with …world events and with the implications of Nietzsche’s inaugural annunciation of modernity in terms of the death of God” (187), her later oeuvre is dedicated to the same work of soul undertaken by the “secret cult of Night” in Vale Ave. Here, her thematic scope faces two ways: backward to ancient Greek mystery cults and their palingenesic rites and forward to depth psychologists searching for the Soul of the World. Vale Ave plays a pronounced role in my study as symbolic guide; in its seventy-four sequences the layering of time in the “trilogy” of past, present, and future that H.D. had explored during the years of the Second World War in order to get behind the fallen walls of cause and effect collapses into two distinct phases of human origin—“meeting” (evolution) and “parting” (involution)—and the poem invites Lilith and Lucifer to be its archetypal guides. My method for the study is imaginal, entering such disciplines as history, philosophy, and theology and bringing psychological understanding to them. John Walsh’s introduction to Vale Ave notes H.D.’s theme “that the human psyche exists in a dimension outside of time and space as well as within them. In Vale Ave, H.D. presents the extremity of this dual-dimensionality: metempsychosis” (vii). However, the concept that H.D. investigates is more than a literary processus of characters who adopt different masks and appear at various junctures in a chronological unwinding of history. I explore H.D.'s works as part of a Modernist tradition of writing “books of the dead” designed not to guide the soul after death, but to draw the gaze upon “a nearer thing,” as H.D. writes in Erige Cor Tuum Ad Me In Caelum, the wisdom intrinsic in the spirit of life itself.