Localization and potential function of kindlin-1 in periodontal tissues

Abstract

Kindlin-1 is an intracellular focal adhesion protein that regulates the actin cytoskeleton. Patients suffering from Kindler syndrome have a homologous mutation of the kindlin-1 gene and develop skin blisters, periodontal disease, and intestinal complications because of deficient adhesion of the basal epithelial cells. We investigated kindlin-1 localization in periodontal tissue and its functions in cultured keratinocytes and showed that kindlin-1 co-localizes with migfilin and paxillin in the basal epithelial cells of oral mucosa and in cultured keratinocytes. The kindlin-1-deficient oral mucosal tissue from a patient with Kindler syndrome showed a complete lack of paxillin and reduced migfilin immunostaining in the basal keratinocytes. Co-immunoprecipitation showed that migfilin directly interacted with kindlin-1. RNA interference-induced kindlin-1 deficiency in keratinocytes led to an altered distribution of migfilin-containing focal adhesions, reduced cell spreading, decreased cell proliferation, and decelerated cell migration. Disruption of microtubules in the kindlin-1-deficient cells further reduced cell spreading, suggesting that microtubules can partially compensate for kindlin-1 deficiency. Kindlin-1 supported mature cell-extracellular matrix adhesions of keratinocytes, as downregulation of kindlin-1 expression significantly reduced the cell-adhesion strength. In summary, kindlin-1 interacts with migfilin and plays a crucial role in actin-dependent keratinocyte cell adhesion essential for epidermal and periodontal health.

Fig. 1

Immunolocalization (VIP Staining) of ILK-1 (B, D), kindlin-2 (E, G), migfilin (F, H), paxillin (I, K), and kindlin-1 (J, L) in human palatal oral epithelium and connective tissue. Panels A and C represent hematoxylin-eosin stained sections from the corresponding area. A magnified view of the basement membrane zone (BMZ) and basal cell layer is provided in the top-right corner of the left side panels. ILK-1 (B) and kindlin-2 (E) do not localize to the basement membrane zone or the basal cells, but are found in between cells in the suprabasal layers and blood vessels (D and G, respectively; arrows). Migfilin (F), paxillin (I), and kindlin-1 (J) localize strongly to the basement membrane zone (arrowheads) and between cells throughout all layers of the epithelium with only migfilin (H) and paxillin (K) localizing to blood vessels (arrows). Kindlin-1 (L) expression is weak in blood vessels (arrows).

Fig 2

Immunolocalization (VIP Staining) of migfilin (B, D), paxillin (E, G), and kindlin-1 (F, H) in the oral epithelium of normal (B, E, F) or kindlin-1 deficient gingiva (D, G, H). Panels A and C represents hematoxylin-eosin stained sections from the corresponding areas. A magnified view of the BMZ and basal cell layer is provided in the top-right corner. Migfilin and paxillin fail to localize to the BMZ in kindlin-1 deficient gingiva. Arrowheads point to the BMZ and arrows to blood vessels, respectively.

Fig. 3

Immunolocalization of filamentous actin (E, F) and focal adhesion proteins ILK-1 (I, J), kindlin-2 (C, D), migfilin (G, H), and kindlin-1 (K, L) in HaCaT keratinocytes spread for 16 hours in the presence (A, C, E, G, I, K) or absence (B, D, F, H, J, L) of TGF-β1. Secondary antibody alone (A, B) was used as a negative control. A magnified view of the focal adhesions is provided in the bottom-right corner of each panel. TGF-β promotes expression of actin filaments and an increase in the size of ILK-1, kindlin-2, migfilin, and kindlin-1 focal adhesions.

Fig. 4

Interaction of migfilin with kindlin-1. A, GST-kindlin-1 specifically binds to migfilin in human 293 cells. GST-kindlin-1-bound glutathione-Sepharose or control beads were incubated with cell lysates and subjected to Western blotting using anti-migfilin antibody. Lane 1, cell lysate; lane 2, cell lysate incubated with GST-Sepaharose beads; lane 3, cell lysate incubated with GST-kindlin-1 beads. B, Immunolocalization of kindlin-1 (green) at the end of the actin filaments (red) in keratinocytes; C, immunolocalization of migfilin (red) at the end of actin filaments in keratinocytes; D–F, double immunolocalization of kindlin-1 (D, green) and migfilin (E, red) in same focal adhesions (F, orange). Arrows point to the focal adhesions.

Fig. 5

Effect of kindlin-1 knockdown on migfilin and paxillin containing focal adhesions. A, expression of kindlin-1 mRNA after 0–3 days following kindlin-1 siRNA transfection relative to transfection with the control siRNA; B, expression of kindlin-1 protein 1–3 days after transfection with the kindlin-1 siRNA (K1) or irrelevant control siRNA (IR). β-actin was used as a loading control; C, Immunolocalization of migfilin and paxillin containing focal adhesions after transfection with kindlin-1 or IR siRNA; D, average number of migfilin containing focal adhesions per cell after siRNA transfections; E–F, distribution of migfilin (E) and paxillin (F) containing focal adhesions according to the size (0–2 µm, 2–5 µm, 5–100 µm) after siRNA transfection. All histograms show the mean +/− SEM of three parallel experiments (* p<0.05, **p<0.01).

Fig. 6

Effect of kindlin-1 knockdown on keratinocyte cell adhesion. A, attachment of siRNA treated HaCaT keratinocytes on glass coverslips over 24 hours in the presence or absence of 10 ng/ml TGFβ1; B, spreading of siRNA treated keratinocytes on extracellular matrix proteins (type I collagen, laminin10/11 and fibronectin) for 45–60 minutes; C, spreading of siRNA treated keratinocytes on fibronectinin the presence of absence of 1 µM colchicine. Relative spreading percentage to no drug treatment (control, 100%) is shown; D, detachment of established keratinocyte cultures (confluent siRNA treated cells cultured for 4 days) with diluted (1:4) trypsin at various time points (0–25 minutes). Representative result (mean +/− SEM of triplicate samples) from three parallel experiments is shown (**p<0.01; *** p<0.001).

Fig. 7

Effect of kindlin-1 knockdown on keratinocyte proliferation and migration. A, number of viable kindlin-1 (kind siRNA) and control (IR siRNA) siRNA transfected HaCaT keratinocytes relative to the seeding density at 3 hours. Representative result from three parallel experiments is shown; B, migration of kindlin-1 and IR siRNA transfected HaCaT keratinocytes for 24 hours into the wounds made by scratching with pipette tip. The number of cells migrated into the wound per unit area was calculated. The histogram shows the relative mean +/− SEM of three parallel experiments (***p<0.0001).