Expression of keratinocyte growth factor and its receptor in rat tracheal cartilage: possible involvement in wound healing of the damaged cartilage

Abstract

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.

Keywords: keratinocyte growth factor; tracheal cartilage; wound repair.

Fig. 1

Photograph of the operative field. Arrow: longitudinal tracheal incision. Arrowhead: three interrupted sutures closing incision.

Fig. 2

Western blot analysis of KGF in adult rat cartilage. Molecular marker (lane 1), lysate of rat tracheal cartilage (lane 2, 20 µg), lysate of rat nasal septum cartilage (lane 3, 20 µg) and lysate of cultured rat nasal septum chondrocytes (lane 4, 20 µg) were separated on a 15–25% gel. A major band corresponding to 28 kD (lanes 2–4) was detected with anti-KGF antibody (arrow).

Fig. 3

Immunohistochemical localization of KGF and KGFR in serial paraffin sections from normal mature rat cartilage. (A) KGF immunoreactivity in rat tracheal cartilage. Arrow: KGF positive chondrocytes. Arrowhead: KGF positive perichondrial cells. (B) KGFR immunoreactivity in rat tracheal cartilage. Arrow: KGFR positive chondrocytes. Arrowhead: KGFR negative perichondrial cells. p: perichondrial area, c: chondrocyte area. Magnification×100. Bars=200 µm and 30 µm (inset in A).

Fig. 4

Serial changes in the histology in paraffin sections of immature and mature post-operative rat trachea. (A) H&E staining on day 5 after operation in the immature rat group. (B) H&E staining on day 28 in the immature rat group. (C) H&E staining on day 5 in the mature rat group. (D) H&E staining on day 28 in the mature rat group. p: perichondrial area, c: chondrocyte area, f: proliferating fibroblast area. Magnification×100. Bar=200 µm.

Fig. 5

Serial changes in the expression of PCNA in paraffin sections of immature and mature post-operative rat trachea. (A) Immunohistochemical detection of PCNA on day 5 in the immature rat group. (B) Immunohistochemical detection of PCNA on day 5 in the mature rat group. p: perichondrial area, c: chondrocyte area. Magnification×100. Bars=200 µm and 30 µm (inset in A, B).

Fig. 6

Immunohistochemical detection of KGF and KGFR in paraffin sections of post-operative rat trachea. (A) Immunohistochemical detection of KGF on day 5 in the immature rat group. (B) Immunohistochemical detection of KGFR on day 5 in the immature rat group. (C) Immunohistochemical detection of KGF on day 5 in the mature rat group. (D) Immunohistochemical detection of KGFR on day 5 in the mature rat group. Arrowhead: positive staining of KGFR in proliferating chondrocytes from perichondrium. p: perichondrial area, c: chondrocyte area. Magnification×100. Bars=200 µm and 30 µm (inset in A, B, C, D).

Fig. 7

Immunohistochemical detection of KGF and KGFR in primary cultures of rat nasal septum chondrocytes. (A) KGF immunoreactivity was detected in primary cultures of rat nasal septum chondrocytes. (B) KGFR immunoreactivity was detected in primary cultures of rat nasal septum chondrocytes. Magnification×400. Bar=50 µm.

Fig. 8

Total cell counts and quantification of acid mucopolysaccharide product in primary cultured chondrocytes treated with various concentrations of KGF. (A) Total cell counts of primary cultured chondrocytes treated with various concentrations of KGF on day 6 during culture. Data are mean±SD of three independent experiments. * P<0.05 vs. control. (B) Quantification of acid mucopolysaccharide product in primary cultured chondrocytes treated with various concentrations of KGF on day 6 during culture. (Product amount per 1000 cells). Data are mean±SD of three independent experiments. * P<0.05 vs. control.