Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein

Abstract

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.

Figure 1

Photographs depicting one of our patients with Costello syndrome at 6 mo of age, whose fibroblasts were used in the presented studies. A, sparse and curly hair, “coarse” face, depressed nasal bridge, bulbous and upturned nose, low-set ears, full cheeks, large tongue, pouting lower lip, strabismus. B, deep palmar creases. C, plantar creases.

Figure 2

Representative photomicrographs of 10-d-old cultures immunostained with antitropoelastin antibody indicate that normal fibroblasts (A) produced long, branching elastic fibers, whereas fibroblasts from patient with Costello syndrome (B) did not deposit any extracellular elastin. Deposition of fibrillin I by Costello fibroblasts (C) does not differ from normal fibroblasts (D). Costello fibroblasts, however, deposit more collagen type 1 (F) and fibronectin (H) than do normal fibroblasts (E and G, respectively). Whereas Costello fibroblasts cultured for 10 d in normal medium did not display any extracellular elastic fibers (I), treatment with chondroitinase ABC (J) restored their ability to deposit elastic fibers in the extracellular matrix.

Figure 3

Morphometric analysis of extracellular matrix (ECM) components immunostained with specific antibodies in 10-d-old cultures of normal and Costello fibroblasts. Fibroblasts from patients with Costello syndrome deposit only negligible amounts of immunodetectable extracellular elastin. Amounts of fibronectin and collagen type I produced by Costello fibroblasts significantly exceed those present in cultures of normal fibroblasts, whereas deposition of fibrillin I and MAGP by Costello fibroblasts does not differ from normal fibroblasts. In each analyzed group, 50 low-power fields (´20) from three separate cultures (per independent patients) were analyzed and the area occupied by the particular immunodetectable component quantified. The abundance of each component was then expressed as a percentage of the entire analyzed field (mean ± SD), and results from cultures of Costello fibroblasts were statistically compared with those in cultures of normal skin fibroblasts (P<.001).

Figure 4

A, Quantitative analysis of [³H]-valine-labeled immunoprecipitatable tropoelastin indicates that both normal (3858, 4184, and 4212) and Costello fibroblasts (7669, 9951, and 10595) synthesize comparable amounts of total metabolically labeled tropoelastin. In contrast to normal fibroblasts, Costello fibroblasts retain the majority of their metabolically labeled tropoelastin intracellularly. Consistently, Costello fibroblasts incorporate much less [³H]-valine into extracellular insoluble elastin than do normal fibroblasts. B, Representative autoradiographs of metabolically radiolabeled proteins immunoprecipitated with antitropoelastin antibody from the cell-layer extracts. In contrast to normal fibroblasts (3858, 4184, and 4212) that contain a significant amount of radiolabeled 70-kD tropoelastin, the Costello fibroblasts (7669, 9951, and 10595) show much lower levels of intact 70-kD tropoelastin and show numerous lower molecular weight species that likely represent tropoelastin degradation products.

Figure 5

A–D, Representative photomicrographs of 48-h-old cultures of lightly fixed and nonpermeabilized fibroblasts. Immunostaining with anti-S-Gal antibody, which recognizes EBP, indicates that normal fibroblasts (A) show strong cell-surface expression of this protein, whereas Costello fibroblasts (B) show greatly diminished levels of the EBP. Immunostaining with monoclonal antibody recognizing chondroitin sulfate A indicates that, in contrast to cultures of normal fibroblasts (C), which show immunodetectable epitope only in the extracellular matrix, Costello fibroblasts show strong cell surface–associated expression of chondroitin sulfate (D). E–J, Representative photomicrographs of 48-h-old cultures of permeabilized fibroblasts immunostained with antibodies to chondroitin sulfate A epitope (E, F) to CD44 (G, H) and to biglycan (I, J). In contrast to normal fibroblasts (E, G, I), which do not show any intracellular immunostaining with anti-CD44 and antibiglycan antibodies, and only perinuclear localization of the chondroitin sulfate epitope, Costello fibroblasts (F, H, J) show strong and overlapping intracellular (lysosomal) localization of all three epitopes.

Figure 6

Representative autoradiographs (upper panel) showing levels of the 67-kD EBP during the chase after metabolic pulse labeling with radioactive serine. Results indicate that both normal and Costello fibroblasts initially synthesize comparable amounts of EBP, isolated by elastin affinity columns. In contrast to normal fibroblasts, which retain the majority of the labeled EBP, Costello fibroblasts steadily lose this newly produced protein, which can be immunoprecipitated in increased amounts from the respective conditioned media during the chase (lower panel) .

Figure 7

A, Representative phase-contrast micrographs of 3-d-old cultures illustrating that the growth rate of fibroblasts from patients with Costello syndrome (middle panel) were considerably higher than those taken from normal children (left panel). Addition of insoluble elastin to cultures of Costello fibroblasts substantially reduced their cell density (right panel). Arrows indicate clusters of insoluble elastin attached to the cultured fibroblast. B, Cell counting and C, incorporation of [³H]-thymidine also show the increased growth rate of Costello fibroblasts (blackened bars) as compared with normal fibroblasts (unblackened bars) in 3-d-old cultures. These assays also illustrate that high proliferation of Costello fibroblasts was significantly reduced in cultures treated with exogenous insoluble elastin (striated bars). Treatment with insoluble elastin did not change the proliferative rate of normal fibroblasts. D, Parallel cultures of normal fibroblasts treated with chondroitin sulfate (CS) or dermatan sulfate (DS), but not heparan sulfate (HS) (all in concentration 400 μg/ml), significantly increased the rate of their [³H]-thymidine incorporation. At the same time, addition of chondroitinase ABC (0.2 U/d) (Chnase ABC) significantly decreased incorporation of [³H]-thymidine in cultures of fibroblasts taken from patients with Costello syndrome. In all experiments, cells were plated with the same initial density of 50,000 cells/well. Values of mean ± SD from three different experiments were statistically compared with untreated controls within the same cell type (P<.002).