Cellular response of cardiac fibroblasts to amyloidogenic light chains

Abstract

Amyloidoses are a group of disorders characterized by abnormal folding of proteins that impair organ function. We investigated the cellular response of primary cardiac fibroblasts to amyloidogenic light chains and determined the corresponding change in proteoglycan expression and localization. The cellular response to 11 urinary immunoglobulin light chains of kappa1, lambda6, and lambda 3 subtypes was evaluated. The localization of the light chains was monitored by conjugating them to Oregon Green 488 and performing live cell confocal microscopy. Sulfation of the proteoglycans was determined after elution over Q1-columns with a single-step salt gradient (1.5 mol/L NaCl) via dimethylmethylene blue. Light chains were detected inside cells within 4 hours and demonstrated perinuclear localization. Over 80% of the cells showed intracellular localization of the amyloid light chains. The light chains induced sulfation of the secreted glycosaminoglycans, but the cell fraction possessed only minimal sulfation. Furthermore, the light chains caused a translocation of heparan sulfate proteoglycan to the nucleus. The conformation and thermal stability of light chains was altered when they were incubated in the presence of heparan sulfate and destabilization of the amyloid light chains was detected. These studies indicate that internalization of the light chains mediates the expression and localization of heparan sulfate proteoglycans.

Figure 1

Light chains conjugated to Oregon Green 488 are localized within cells. Cardiac fibroblasts were cultured for 4 hours in the presence of AL LC κ1 conjugated to Oregon Green 488 and imaged live using confocal microscopy. A Z-series of 1-μm optical sections was taken through cells. Arrows indicate light chain present in middle optical sections. Images are representative of a minimum of five experiments with independent conjugations.

Figure 2

Long-term incubation (5 days) with AL - κ1 and λ6-LCs induces a filamentous appearance in AL LCs (AL-96066, AL-01066, AL-01029). Cultures were fixed and stained with rhodamine-phalloidin to detect F-actin. A–D: A, κ1; B, κ1; C, λ6; and D, non-AL κ1. Composite images do not show co-localization of F-actin and LC. Images are representative of a minimum of five independent experiments.

Figure 3

Nuclear localization of HSPG 24 hours after LC (unconjugated) was added to cell and stained for HSPG with the mAb Δ3G10. Cells were fixed, treated with heparinase III, and stained with Δ3G10 and rhodamine-phalloidin. A and B: The addition of AL LC κ1 (AL-96066) shows HSPG in the nucleus of confluent and in semi-confluent cells (arrows). C: The addition of non-AL LC κ1 (MM-96100) does not induce translocation of HSPG. D: The addition of AL LC κ1 (AL-96066) to cells that were treated with heparinase III 1 hour before fixation and then stained for Δ3G10 shows HSPG on the cell margins (arrowhead). Images are representative of five independent experiments.

Figure 4

AL LCs induce remodeling of cellular HSPGs in long-term cultures. Confocal images of cells. Cells were incubated with Oregon Green 488 conjugated to a specific light chain for 7 days, fixed, permeabilized, treated with heparinase III, and stained with mAb Δ3G10 and rhodamine phalloidin. A Z-series of 1-μm optical sections was taken through the cell cultures from the basal to the apical surface of the cell and composites were generated using LSM 5 Image software. A: Cells incubated in the presence of an AL LC κ1 conjugated to Oregon Green 488. Orthogonal slice analysis was performed on the composite using LSM 510 Image software (XZ is depicted on the horizontal and YZ on the vertical axis outside the dimensions of the composite). Co-localization of HSPG and LC is demonstrated. Optical sections of the composite show three regions within the cell and demonstrate localization of HSPG, LC, and F-actin in these regions. HSPG is most prominent in the apical optical section and LC is detected in the central region. B: Cell cultures incubated with non-AL LC κ1 conjugated to Oregon Green 488 for 7 days. Cells were stained and fixed as described above. HSPG is negligible in comparison to A. Images are representative of a minimum of three independent experiments.

Figure 5

FGF-2 and LCs mediate the sulfation of secreted and cellular GAGs. A and B: Representative profiles of media (A) and cell elution profiles (B) from Q1-Sepharose with 1.5 mol/L NaCl single-step gradient. Elution profile is representative of three independent experiments and of nine AL LCs evaluated.

Figure 6

Syndecan-1 and glypican-1 in response to AL LCs. Twelve percent SDS-PAGE and Western blot of media, matrix and cell lysates immunoblotted with MAbs to syndecan-1, and glypican-1. Equivalent amounts of protein were loaded on each lane. Lanes 1–4: Cell; 5–8, Medium; 9–12, ECM. Lanes representing cells treated with AL LC κ1 (AL-00131) 3–4, 7–8, and 11–12. Lanes representing treatment with Heparinase I/III (1.0/2.0U/ml) and Case ABC (0.5U/ml), Cell, 2 and 4; Medium, 6 and 8; Matrix, 10 and 12. Control lanes (1–2, 5–6, 9–10).

Figure 7

Heparan sulfate alters the Far UV spectra and the thermal unfolding of 2 LC κ 1s. Far UV CD Spectra of AL LC κ1 (AL-96066) (A), and AL LC κ1 (AL-00131) (B) with and without HS in PBS at pH 7.4 were recorded at 25°C from 250 to 200 nm. Thermal unfolding of AL-96066 (C) and AL-00131 (D) LCs with and without HS in PBS at pH 7.4 monitored at 217 nm (β-sheet). Ellipticity was continuously monitored from 5 to 95°C every 0.5°C with an averaging time of 90 seconds. Protein concentration, 0.40 mg/ml. LC: HS, 4:1 w/w; pathlength, 0.05 cm.