Faculty Bibliography

Cellular interaction with the extracellular matrix is thought to be a critical event in controlling angiogenesis and tumor growth. In our previous studies, genetically distinct noncollagenous (NC) domains of type-IV collagen were shown to interact with integrin receptors expressed on the surface of endothelial cells. Moreover, these NC1 domains were shown to inhibit angiogenesis in vivo. Here, we provide evidence that a recombinant form of the alpha2(IV)NC1 domain of type-IV collagen could bind integrins alpha1beta1 and alphavbeta3 expressed on melanoma cells and inhibit tumor cell adhesion in a ligand-specific manner. Systemic administration of recombinant alpha2(IV)NC1 domain potently inhibited M21 melanoma tumor growth within full thickness human skin and exhibited a dose-dependent inhibition of tumor growth in nude mice. Interestingly, alpha2(IV)NC1 domain enhanced cellular senescence in tumor cells in vitro and in vivo. Taken together, these results suggest that recombinant alpha2(IV)NC1 domain is not only a potent anti-angiogenic reagent, but it also directly impacts tumor cell behavior. Thus, alpha2(IV)NC1 domain represents a potent inhibitor of tumor growth by impacting both endothelial and tumor cell compartments

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.

Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

The process of implantation absolutely requires synchronized development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a reciprocal dialogue between the blastocyst and uterine luminal epithelium. Genetic and molecular approaches have identified several signaling pathways that are critical to this process. The transcription factor Hoxa10 is one such critical player in implantation. Hoxa10-/- female mice have implantation and decidualization failure due specifically to reduced uterine responsiveness to progesterone and defective stromal cell proliferation during uterine receptivity and implantation. However, the downstream signaling pathways of Hoxa10 in these events remain largely unknown. Using the proteomics approach of difference gel electrophoresis, we have identified an immunophilin FKBP52 (FK506 binding protein 4) as one of the Hoxa10-mediated signaling molecules in the uterus. We found that FKBP52, a cochaperone protein known to influence steroid hormone receptor functions, is down-regulated in stromal cells of Hoxa10-/- mice. More importantly, FKBP52 shows differential uterine cell-specific expression during the periimplantation period. Whereas it is primarily expressed in the uterine epithelium on d 1 of pregnancy, the expression expands to the stroma on d 4 during the period of uterine receptivity and becomes localized to decidualizing stromal cells surrounding the implantation site on d 5. This suggests that FKBP52 is important for the attainment of uterine receptivity and implantation. Furthermore, FKBP52 shows differential cell-specific expression in the uterus in response to progesterone and/or estrogen consistent with its expression patterns during the periimplantation period. Collectively, these results and the female infertility phenotype of FKBP52 suggest that a Hoxa10-FKBP52 signaling axis is critical to uterine receptivity and implantation.

Real-space refinement has been previously introduced as a flexible fitting method to interpret medium-resolution cryo-EM density maps in terms of atomic structures. In this way, conformational changes related to functional processes can be analyzed on the molecular level. In the application of the technique to the ribosome, quasiatomic models have been derived that have advanced our understanding of translocation. In this article, the choice of parameters for the fitting procedure is discussed. The quality of the fitting depends critically on the number of rigid pieces into which the model is divided. Suitable quality indicators are crosscorrelation, R factor, and density residual, all of which can also be locally applied. The example of the ribosome may provide some guidelines for general applications of real-space refinement to flexible fitting problems

Turbid growth of some Candida albicans isolates occurs, paradoxically, in some high concentrations of caspofungin, above the minimum inhibitory concentration. We show that the resistant phenotype is first detectable after 24 h of drug exposure. Although other studies have suggested an association between some azole resistance mechanisms and caspofungin resistance, our studies with isolates susceptible and resistant to azoles (the latter including groups with defined resistance mechanisms and derived mutants) suggest a weak association at most with a paradoxical effect. The paradoxical growth is not related to mutations in resistance-associated regions of the (1,3)-beta-glucan synthase complex and is not related to an up-regulation of (1,3)-beta-glucan synthase activity in the presence of drug. Subculture of a minority of tubes above the minimum fungicidal concentration yielded a few viable cells, suggesting random distribution, in some strains, of a few cells with propensity to grow in the presence of drug. We postulate high drug concentrations derepress or activate an as-yet undefined resistance mechanism(s).

Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids

Experiments using mammalian epithelial cell lines have elucidated biosynthetic and recycling pathways for apical and basolateral plasma-membrane proteins, and have identified components that guide apical and basolateral proteins along these pathways. These components include apical and basolateral sorting signals, adaptors for basolateral signals, and docking and fusion proteins for vesicular trafficking. Recent live-cell-imaging studies provide a real-time view of sorting processes in epithelial cells, including key roles for actin, microtubules and motors in the organization of post-Golgi trafficking.