ERK activation is required for CCK-mediated pancreatic adaptive growth in mice.

High levels of cholecystokinin (CCK) can stimulate pancreatic adaptive growth in which mature acinar cells divide, leading to enhanced pancreatic mass with parallel increases in protein, DNA, RNA, and digestive enzyme content. Prolonged release of CCK can be induced by feeding trypsin inhibitor (TI) to disrupt normal feedback control. This leads to exocrine growth in a CCK-dependent manner. The extracellular signal-related kinase (ERK) pathway regulates many proliferative processes in various tissues and disease models. The aim of this study was to evaluate the role of ERK signaling in pancreatic adaptive growth using the MEK inhibitors PD-0325901 and trametinib (GSK-1120212). It was determined that PD-0325901 given two times daily by gavage or mixed into powdered chow was an effective and specific inhibitor of ERK signaling in vivo. TI-containing chow led to a robust increase in pancreatic mass, protein, DNA, and RNA content. This pancreatic adaptive growth was blocked in mice fed chow containing the MEK inhibitors. PD-0325901 blocked TI-induced ERK-regulated early response genes, cell-cycle proteins, and mitogenesis by acinar cells. It was determined that ERK signaling is necessary for the initiation of pancreatic adaptive growth but not necessary to maintain it. PD-0325901 blocked adaptive growth when given before cell-cycle initiation but not after mitogenesis had been established. Furthermore, GSK-1120212, a chemically distinct inhibitor of the ERK pathway that is now approved for clinical use, inhibited growth similar to PD-0325901. These data demonstrate that the ERK pathway is required for CCK-stimulated pancreatic adaptive growth.

Quantitative glycomics from fluidic glycan microarrays.

A hallmark of cell-surface processes involving glycans is their multivalent interaction with glycan binding proteins (GBPs). Such a multivalent interaction depends critically on the mobility and density of signaling molecules on the membrane surface. While glycan microarrays have been used in exploring multivalent interactions, the lack of mobility and the difficulty in controlling surface density both limit their quantitative applications. Here we apply a fluidic glycan microarray, with glycan density varying for orders of magnitude, to profile cell surface interaction using a model system, the adhesion of Escherichia coli to mannose. We show the quantitative determination of monovalent and multivalent adhesion channels; the latter can be inhibited by nanopartices presenting a high density of mannosyl groups. These results reveal a new E. coli adhesion mechanism: the switching in the FimH adhesion protein avidity from monovalent to multivalent as the density of mobile mannosyl groups increases; such avidity switching enhances binding affinity and triggers multiple fimbriae anchoring. Affinity enhancement toward FimH has only been observed before for oligo-mannose due to the turn on of secondary interactions outside the mannose binding pocket. We suggest that the new mechanism revealed by the fluidic microarray is of general significance to cell surface interactions: the dynamic clustering of simple sugar groups (homogeneous or heterogeneous) on the fluidic membrane surface may simulate the functions of complex glycan molecules.

Fluidic and air-stable supported lipid bilayer and cell-mimicking microarrays.

As drug delivery, therapy, and medical imaging are becoming increasingly cell-specific, there is a critical need for high fidelity and high-throughput screening methods for cell surface interactions. Cell membrane-mimicking surfaces, i.e., supported lipid bilayers (SLBs), are currently not sufficiently robust to meet this need. Here we describe a method of forming fluidic and air-stable SLBs through tethered and dispersed cholesterol groups incorporated into the bottom leaflet. Achieving air stability allows us to easily fabricate SLB microarrays from direct robotic spotting of vesicle solutions. We demonstrate their application as cell membrane-mimicking microarrays by reconstituting peripheral as well as integral membrane components that can be recognized by their respective targets. These demonstrations establish the viability of the fluidic and air-stable SLB platform for generating content microarrays in high throughput studies, e.g., the screening of drugs and nanomedicine targeting cell surface receptors.

Denaturing and refolding of protein molecules on surfaces.

Keeping protein molecules in the active state on a solid surface is essential to protein microarrays and other protein-based biosensors. Here, we show that the 2-D chemical environment controls the refolding of the denatured green fluorescent proteins tethered to solid surfaces. Refolding occurs readily on the repulsive PEG functionalized surface but is inhibited on the attractive--NH(2) functionalized surface. This result shows the critical importance of the 2-D chemical environment in the maintenance and revival of protein activity on surfaces and opens the door to designing 2-D molecular chaperones for protein folding.