Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4 ؊/؊ mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4 ؊/؊ embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4 ؊/؊ mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4 ؊/؊ tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4 ؊/؊ mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.Elastic fibers with morphologically distinct architectures are present in the extracellular matrix (ECM) to accommodate elastic requirements and mechanical stresses imposed on different tissues. They are particularly abundant in elastic tissues such as large blood vessels, lung, and skin. Loss of elasticity is a major contributing factor in aging and a myriad of pathological conditions including emphysema, artery diseases, and cutis laxa (39,41,44). Elastic fibers undergo irreversible structural and compositional changes with age and in some pathological conditions (41). Regardless of morphology, all elastic fibers consist of cross-linked elastin, fibrillin-rich microfibrils, and several associated molecules (23,37,38,46). Elastin endows the fiber with the characteristic property of elastic recoil. It is chemically inert, extremely hydrophobic, and insoluble under most conditions. Monomeric elastin, called tropoelastin, is secreted from the cell as a soluble protein. Isolated and purified tropoelastin has been shown to exhibit a great tendency to aggregate (coacervation) in physiological solution and at temperatures in the physiological range, giving rise to supramolecular structures very similar to those found in natural elastic fibers (4,5,11). This self-aggregation property of tropoelastin is thought to contribute to elastic fiber assembly in vivo. However, self-aggregation alone is insufficient to explain the efficiency of the assembly process and the variable form of elastic fibers ...
Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.
Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles. Myocilin (MYOC),1 also known as trabecular meshwork inducible glucocorticoid response protein, is an acidic 504-amino acid protein. Structurally, MYOC contains at least two folding domains, an N-terminal coiled-coil and a C-terminal globular domain with significant homology to an olfactomedin module present in several different proteins (1, 2). Mammalian proteins with the olfactomedin module localize to different compartments of the secretory pathway, although little is known about the function of these proteins or the olfactomedin module (3-10).Despite a broad expression pattern (1, 2, 7, 11-13), the function of MYOC remains unknown and its cellular distribution ambiguous. For example, MYOC has been observed in various cell types to associate with structures that are part of the secretory pathway, including endoplasmic reticulum, Golgi apparatus, and intracellular vesicles. Conversely, MYOC has also been reported to associate with mitochondria and cytoplasmic filaments (11, 14 -17). Even more unusual, MYOC appears to be secreted by some cell types, but not by others (2, 18). Thus, MYOC is expressed by retinal ganglion cells, photoreceptors, and retinal pigment epithelium, but is not found extracellularly in the retina nor in conditioned medium of retinal pigment cells in culture (2,18,19). In contrast, MYOC is found in conditioned medium of trabecular meshwork (TM) ...
The expression of AQP1 by RPE in vivo probably contributes to the efficient transepithelial water transport across RPE, maintains retinal attachment, and prevents subretinal edema.
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