Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.

In vitro generation of three-dimensional renal structures.

End-stage renal disease is currently being treated effectively by transplantation. However, increasing demand and donor shortage make this treatment challenging. Recent advances in cell-based therapies have provided potential opportunities to alleviate the current challenges of donor shortage. In this study we developed a system to generate renal structures in vitro using primary kidney cells. This system involves the cultivation of expanded primary renal cells in a three-dimensional collagen-based culture system. After one week of growth, individual renal cells began to form renal structures resembling tubules and glomeruli. Histologically, these structures show phenotypic resemblance to native kidney structures. The reconstituted tubules stained positively for Tamm-Horsfall protein, which is expressed in the thick ascending limb of Henle's Loop and distal convoluted tubules. These results show that renal structures can be reconstituted in a three-dimensional culture system, which may eventually be used for renal cell therapy applications.

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate.

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.

The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo.

Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissue's resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cell's microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.