Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.     

Mast cell tryptase stimulates human lung fibroblast proliferation via protease-activated receptor-2

Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.     

In vivo resolution of multiexponential decays of multiple near-infrared molecular probes by fluorescence lifetime-gated whole-body time-resolved diffuse optical imaging

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.     

Interaction of fluorescent molecular rotors with blood plasma proteins

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Near-infrared fluorescent divalent RGD ligand for integrin α(v)β(3)-targeted optical imaging

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)β(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)β(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.     

Bone marrow osteoblast damage by chemotherapeutic agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.     

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.