Faculty Bibliography

Signaling through Trk receptor tyrosine kinases can occur in the absence of neurotrophins through certain G-protein-coupled receptors (GPCRs). It has previously been suggested that GPCR-mediated Trk activation occurs on intracellular membranes and involves several second messengers, including Src family kinases and intracellular calcium. Here, we describe a novel role for the Src family kinase, Fyn, in regulating signaling events between GPCRs and Trk. We find that Fyn expression is sufficient to allow transactivation of Trk by adenosine and that Fyn and Trk are colocalized in a juxtanuclear membrane compartment. Adenosine activation of Fyn results in direct phosphorylation of Trk in vitro and follows a delayed time course that coincides with Trk activation. These results indicate that Fyn is activated by GPCR stimulation and is responsible for transactivation of Trk receptors on intracellular membranes

The Hedgehog pathway is vital for the development of many epidermal appendages, but its role in mammary development has been unclear. Here, we show that although Gli2 and Gli3 are expressed during embryonic mammary development, transcriptional reporters of positive Hedgehog signaling are absent. Nevertheless, Gli3(xt/xt) embryos show aberrant early mammary marker expression and lack two pairs of mammary buds, demonstrating that Gli3 is essential for mammary bud formation and preceding patterning events. Misactivation of the Hedgehog pathway by targeted expression of the constitutive activator Gli1, from the Gli2 promoter in Gli3(xt/+) mice, also induces mammary bud loss. Moreover, loss of Gli3 expression induces Gli1 misexpression in mammary mesenchyme. These results establish that the essential function of Gli3 during embryonic mammary development is to repress Hedgehog/Gli1-inducible targets. During postnatal mammary development, Gli2 and Gli3 are expressed in stromal and myoepithelial cells, and Gli3 is also found within the lumenal epithelium. Again, transcriptional reporters of positive Hedgehog signaling are absent from these cell types, yet are expressed robustly within mammary lymphatics. Thus, positive Hedgehog signaling is absent throughout mammary development, distinguishing the mammary gland from other epidermal appendages, such as hair follicles, which require Hedgehog pathway activity

The ability to specifically image macrophages may enable improved detection and characterization of atherosclerosis. In this study we evaluated the in vitro uptake of gadolinium (Gd)-containing immunomicelles (micelles linked to macrophage-specific antibody), micelles, and standard contrast agents by murine macrophages, and sought to determine whether immunomicelles and micelles improve ex vivo imaging of apolipoprotein E knockout (ApoE KO) murine atherosclerosis. Murine RAW 264.7 macrophages were incubated with Gd-DTPA, micelles, and immunomicelles. Cell pellets were prepared and imaged using a 1.5 T MR system with an inversion recovery spin-echo sequence to determine the in vitro T1 values. Ex vivo analysis of mouse aortas was performed using a 9.4T MR system with a high-spatial-resolution sequence (78x39x78 microm3). The T1 value was significantly decreased in cells treated with micelles compared to Gd-DTPA (P<0.0001), and in cells incubated at 4 degrees C with immunomicelles compared to micelles (P<0.05). Ex vivo MRI signal intensity (SI) was significantly increased by 81% and 20% in aortas incubated with immunomicelles and micelles, respectively. Confocal microscopy demonstrated in vitro and ex vivo uptake of fluorescent immunomicelles by macrophages. Immunomicelles and micelles improve in vitro and ex vivo MR detection of macrophages, and may prove useful in the detection of macrophage-rich plaques.

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRTare substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production

Autophagy, a lysosomal pathway for degrading organelles and long-lived proteins, is becoming recognized as a key adaptive response that can preclude death in stressed or diseased cells. However, during development strong induction of autophagy in specific cell populations mediates a type of programmed cell death that has distinctive 'autophagic' morphology and a requirement for autophagy activity. The recent identification of autophagosomes in neurons in a growing number of neurodegenerative disorders has, therefore, sparked controversy about whether these structures are contributing to neuronal cell death or protecting against it. Emerging evidence supports the view that induction of autophagy is a neuroprotective response and that inadequate or defective autophagy, rather than excessive autophagy, promotes neuronal cell death in most of these disorders. In this review, we consider possible mechanisms underlying autophagy-associated cell death and their relationship to pathways mediating apoptosis and necrosis.

KvLm, a novel bacterial depolarization-activated K(+) (Kv) channel isolated from the genome of Listeria monocytogenes, contains a voltage sensor module whose sequence deviates considerably from the consensus sequence of a Kv channel sensor in that only three out of eight conserved charged positions are present. Surprisingly, KvLm exhibits the steep dependence of the open channel probability on membrane potential that is characteristic of eukaryotic Kv channels whose sensor sequence approximates the consensus. Here we asked if the KvLm sensor shared a similar fold to that of Shaker, the archetypal eukaryotic Kv channel, by examining if interactions between conserved residues in Shaker known to mediate sensor biogenesis and function were conserved in KvLm. To this end, each of the five non-conserved residues in the KvLm sensor were mutated to their Shaker-like charged residues, and the impact of these mutations on the voltage dependence of activation was assayed by current recordings from excised membrane patches of Escherichia coli spheroplasts expressing the KvLm mutants. Conservation of pairwise interactions was investigated by comparison of the effect of single mutations to the impact of double mutations presumed to restore wild-type fold and voltage sensitivity. We observed significant functional coupling between sites known to interact in Shaker Kv channels, supporting the notion that the KvLm sensor largely retains the fold of its eukaryotic homologue.

The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question--how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras

PURPOSE: This study investigated the presence of the 27-kd heat shock protein (HSP27) and its responses to ultraviolet B (UVB) irradiation in human corneal epithelium and in cultured corneal epithelial cells. METHODS: Human corneal epithelial cells including presumed corneal epithelial stem cells were cultured in vitro. HSP27 expression and intracellular localization in normal corneas or cultured corneal cells were examined using immunofluorescence staining. The expression of HSP27 in cultured corneal cells was also detected using western blotting, and the phosphorylated isoforms of HSP27 were identified using isoelectric focusing. RESULTS: In normal corneal tissue, HSP27 was present in limbal basal and suprabasilar epithelial cells. In cultured epithelial corneal cells, HSP27 expression was heterogeneous: Some cells expressed virtually no HSP27 and others showed relatively strong expression. HSP27 was localized to the cytoplasm in nonstressed cells and translocated to the perinuclear and nuclear areas after UVB irradiation. UVB irradiation also induced the phosphorylation of HSP27, resulting in the increase in monophosphorylated isoform and formation of biphosphorylated isoform. UV induced the phosphorylation of HSP27 apparently through activation of p38 mitogen-activated protein kinase. CONCLUSION: HSP27 is present mainly as a nonphosphorylated isoform in corneal epithelium and cultured corneal epithelial cells under nonstressed conditions. The constitutional expression of HSP27 suggests that it plays a physiologic role in the cornea. After UVB irradiation, HSP27 undergoes rapid phosphorylation and translocation. This stress response may be related to a protective role of HSP27 for survival of UVB-exposed corneal cells

Tissue patterning must be translated into morphogenesis through cell shape changes mediated by remodeling of the actin cytoskeleton. We have found that Capping protein alpha (Cpa) and Capping protein beta (Cpb), which prevent extension of the barbed ends of actin filaments, are specifically required in the wing blade primordium of the Drosophila wing disc. cpa or cpb mutant cells in this region, but not in the remainder of the wing disc, are extruded from the epithelium and undergo apoptosis. Excessive actin filament polymerization is not sufficient to explain this phenotype, as loss of Cofilin or Cyclase-associated protein does not cause cell extrusion or death. Misexpression of Vestigial, the transcription factor that specifies the wing blade, both increases cpa transcription and makes cells dependent on cpa for their maintenance in the epithelium. Our results suggest that Vestigial specifies the cytoskeletal changes that lead to morphogenesis of the adult wing