Fibulin-5 contributes to microfibril assembly in human periodontal ligament cells

Abstract

The elastic system fibers comprise oxytalan, elaunin and elastic fibers, which differ in their relative microfibril and elastin content. Human periodontal ligaments (PDL) contain only oxytalan fibers (pure microfibrils) among them. Since fibulin-5 regulates the organization of elastic fibers to link the fibers to cells, we hypothesized that fibulin-5 may contribute to the formation of oxytalan fibers. We used siRNA for fibulin-5 in PDL cell culture to examine the extracellular deposition of fibrillin-1 and -2, which are the major components of microfibrils. Fibulin-5 was labeled on microfibrils positive for fibrillin-1 and -2. Fibulin-5 suppression reduced the level of fibrillin-1 and -2 deposition to 60% of the control level. These results suggest that fibulin-5 may control the formation of oxytalan fibers, and play a role in the homeostasis of oxytalan fibers.

Keywords: fibrillin; fibulin-5; microfibrils; oxytalan fiber; periodontal ligaments.

Fig. 1

Characterization of anti-fibrillin-1, fibrillin-2 and fibulin-5 antibodies. (A) Immunoblotting of PDL culture medium against anti-fibrillin-1 (left lane), anti-fibrillin-2 (middle lane) and anti-fibulin-5 (right lane) antibodies. Fibrilin-1, fibrillin-2 and fibulin-5 antibodies interact with an estimated single protein. (B) Immunoblotting against anti-fibrillin-1 (left lane) and anti-fibulin-5 (right lane) antibodies following immunoprecipitation of PDL culture medium using anti-fibrillin-1 antibody. The immunoprecipitates were reasonably recognized by fibrillin-1 antibody (arrowhead), and the fibulin-5 antibody did not cross-react with the immunoprecipitates. (C) Immunoblotting against anti-fibrillin-1 (left lane), anti-fibrillin-2 (middle lane) and anti-fibulin-5 (right lane) antibodies following immunoprecipitation using anti-fibulin-5 antibody. The immunoprecipitates were reasonably recognized by fibulin-5 antibody (arrow), and neither of the other two antibodies cross-reacted with the immunoprecipitates.

Fig. 2

Immunolocalization of fibulin-5 to microfibrils. Double immunofluorescence of fibrillin-1/fibulin-5 and fibrillin-2/fibulin-5 in PDL fibroblasts cultures. Human PDL fibroblasts were cultured for 14 days, then simultaneously labeled with fibrillin-1 (green), fibulin-5 (red), and superimposition of both labels (upper panels). Similarly, the cells were labeled with fibrillin-2 (green), fibulin-5 (red) and superimposition (lower panels). Bar=100 µm.

Fig. 3

Fibulin-5 siRNA suppresses deposition of its protein. PDL fibroblasts were cultured for 7 days, and then transiently transfected with 0, 50, 200 nM fibulin-5 siRNA for another 7 days. (A) cell/matrix lysates (5 µg) were analyzed by western blotting. Mock-transfected (vehicle only) culture was used as a control (second lane). A549 cell lysates were used as fibulin-5-positive controls (first lane). β-actin was used as an internal control in each lane. (B) PDL fibroblasts were immunolabeled with anti-fibulin-5 antibody (red). SYTOX® Green was used for nuclear staining (green). Bar=100 µm.

Fig. 4

Immunodetection of fibrillin-1 and fibrillin-2 in cell/matrix samples from PDL fibroblasts. PDL fibroblasts were transiently mock-transfected (lane 1), or transiently transfected with 200 nM siRNAs for fibulin-5 (lane 2). (A) Western blots of matrix and cellular samples of PDL fibroblasts cultured for 14 days. Equal amounts of proteins (5 µg) were separated by SDS-PAGE and transferred to Immobilon-P membranes. The blots were probed with anti-fibrillin-1 and anti-fibrillin-2 antibodies. Each antibody recognized a 350-kDa band (arrow). Densitometric analysis of changes in the levels of matrix and cell samples of fibrillins-1 and -2 was conducted using Image J software, and the value for each control sample were arbitrarily assigned a value of 100. The results are representative of three independent experiments. Data are presented as the mean±standard deviation (*p<0.05). (B) Northern blots of RNA samples (1 µg) extracted from PDL fibroblasts at 14 days. (C) Immunofluorescence staining was performed with anti-fibrillin-1 antibody (upper panels) and anti-fibrillin-2 antibody (lower panels). SYTOX® Green was used for nuclear staining (green). Bar=100 µm.