Colin Jamora

Sticky Business: Orchestrating Cellular Signals at Adherens Junctions

Cohesive sheets of epithelial cells are a fundamental feature of multicellular organisms and are largely a product of the varied functions of adherens junctions. These junctions and their cytoskeletal associations contribute heavily to the distinct shapes, polarity, spatially oriented mitotic spindle planes, and cellular movements of developing tissues. Deciphering the underlying mechanisms that govern these conserved cellular rearrangements is a prerequisite to understanding vertebrate morphogenesis.

Intercellular adhesion, signalling and the cytoskeleton

Connections between the cytoskeleton and intercellular junctions profoundly influence cell shape and motility. It is becoming increasingly clear that in addition to structural functions, components of the adhesion apparatus also possess signalling capabilities. Recent studies suggest that their dual function may provide the means to integrate changes in morphology and gene expression during tissue and organ development.

Links between signal transduction, transcription and adhesion in epithelial bud development

The morphogenesis of organs as diverse as lungs, teeth and hair follicles is initiated by a downgrowth from a layer of epithelial stem cells. During follicular morphogenesis, stem cells form this bud structure by changing their polarity and cell–cell contacts. Here we show that this process is achieved through simultaneous receipt of two external signals: a Wnt protein to stabilize β-catenin, and a bone morphogenetic protein (BMP) inhibitor to produce Lef1. β-Catenin then binds to, and activates, Lef1 transcription complexes that appear to act uncharacteristically by downregulating the gene encoding E-cadherin, an important component of polarity and intercellular adhesion. When either signal is missing, functional Lef1 complexes are not made, and E-cadherin downregulation and follicle morphogenesis are impaired. In Drosophila, E-cadherin can influence the plane of cell division and cytoskeletal dynamics. Consistent with this notion, we show that forced elevation of E-cadherin levels block invagination and follicle production. Our findings reveal an intricate molecular programme that links two extracellular signalling pathways to the formation of a nuclear transcription factor that acts on target genes to remodel cellular junctions and permit follicle formation.

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

Abstract We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

At the Roots of a Never-Ending Cycle

Recent studies have yielded a number of important insights into the mechanisms of hair follicle development and cycling and have highlighted the particularly important roles played by stem cells and Wnt signaling pathways.

Epidermal Sensing of Oxygen Is Essential for Systemic Hypoxic Response

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

Regulation of Golgi Structure through Heterotrimeric G Proteins

We have previously shown that ilimaquinone (IQ), a marine sponge metabolite, causes complete vesiculation of the Golgi stacks. By reconstituting the IQ-mediated vesiculation of the Golgi apparatus in permeabilized cells, we now demonstrate that this process does not require ARF and coatomers, which are necessary for the formation of Golgi-derived COPI vesicles. We find that IQ-mediated Golgi vesiculation is inhibited by G alpha(s)-GDP and G alpha(i3)-GDP. Interestingly, adding betagamma subunits in the absence of IQ is sufficient to vesiculate Golgi stacks. Our findings reveal that IQ-mediated Golgi vesiculation occurs through activation of heterotrimeric G proteins and that it is the free betagamma, and not the activated alpha subunit, that triggers Golgi vesiculation.

A signaling pathway involving TGF-β2 and Snail in hair follicle morphogenesis

In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis, Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.

Dynamic expression of epidermal caspase 8 simulates a wound healing response

Tissue homeostasis and regeneration are regulated by an intricate balance of seemingly competing processes—proliferation versus differentiation, and cell death versus survival. Here we demonstrate that the loss of epidermal caspase 8, an important mediator of apoptosis, recapitulates several phases of a wound healing response in the mouse. The epidermal hyperplasia in the caspase 8 null skin is the culmination of signals exchanged between epidermal keratinocytes, dermal fibroblasts and leukocytic cells. This reciprocal interaction is initiated by the paracrine signalling of interleukin 1α (IL1α), which activates both skin stem cell proliferation and cutaneous inflammation. The non-canonical secretion of IL1α is induced by a p38-MAPK-mediated upregulation of NALP3 (also known as NLRP3), leading to inflammasome assembly and caspase 1 activation. Notably, the increased proliferation of basal keratinocytes is counterbalanced by the growth arrest of suprabasal keratinocytes in the stratified epidermis by IL1α-dependent NFκB signalling. Altogether, our findings illustrate how the loss of caspase 8 can affect more than programmed cell death to alter the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.