Fibromodulin Expression in Folliculostellate Cells and Pericytes Is Promoted by TGFβ Signaling in Rat Anterior Pituitary Gland

Abstract

Fibromodulin belongs to the family of small leucine-rich proteoglycans (SLRPs), an active component of extracellular matrix. It directly binds collagens to promote fibrillogenesis and also binds transforming growth factor-beta (TGFβ) to antagonize its actions. Our previous studies of rat anterior pituitary gland revealed that fibromodulin is expressed in folliculostellate cells and pericytes. Although our recent study showed that TGFβ2 secreted from folliculostellate cells induces collagen synthesis in pericytes, the involvement of fibromodulin in TGFβ2-mediated collagen regulation has not been studied. The present study examined the effect of TGFβ2 on fibromodulin synthesis in rat anterior pituitary gland. In situ hybridization for TGFβ receptor II and immunohistological techniques revealed the presence of TGFβ receptor II in folliculostellate cells and pericytes. To confirm canonical TGFβ intracellular signaling, Smad2 immunocytochemistry was performed. Nuclear translocation of Smad2 was observed in folliculostellate cells and pericytes after TGFβ2 treatment. TGFβ2 strongly enhanced fibromodulin mRNA and protein expressions, and TGFβ2-induced mRNA expression was completely blocked by TGFβ receptor I inhibitor (SB431542). These results suggest that folliculostellate cells and pericytes exhibit canonical TGFβ2 signaling, which is associated with fibromodulin production. Thus, this is the first report to show that TGFβ signaling regulates the endogenous TGFβ antagonist fibromodulin in the gland.

Keywords: fibromodulin; folliculostellate cell; pericyte; small leucine-rich proteoglycans; transforming growth factor beta.

Fig. 1.

Expression of TGFβ receptor II mRNA in folliculostellate cells and pericytes of rat anterior pituitary gland. The first row panels show hematoxylin and eosin staining of a cryosection of rat anterior pituitary gland (a) and in situ hybridization of the TGFβ receptor II antisense probe (b). The second row panels show images of in situ hybridization of TGFβ receptor II (c: antisense, d: sense) in the anterior lobe. TGFβ receptor II-expressing cells were observed in parenchymal cells (arrowhead), perivascular cells (arrows), and endothelial cells (double arrowheads). The third row panels show immunohistochemistry of S100 protein (e) and desmin (f), combined with TGFβ receptor II in situ hybridization. The magnified images of (e) and (f) are shown in (g) and (h), respectively. S100 protein was used as a marker for folliculostellate cells, and desmin was used as a marker for pericytes. Immunohistochemical and in situ hybridization signals are shown in brown and purple, respectively. S100 protein-cells (g: open arrow) and desmin-positive cells (h: open arrowhead) expressed TGFβ receptor II. AL: anterior lobe, IL: intermediate lobe, PL: posterior lobe. Bars=100 μm (a, b, e, and f) and 10 μm (c, d, g, and h). Asterisks: capillary lumen.

Fig. 2.

Detection of Smad signaling in folliculostellate cells and pericytes. Anterior pituitary cells from S100β-GFP transgenic rats (A) or Wistar rats (B) were treated with TGFβ2 (50 ng/ml) or 0.1% BSA (vehicle) for 30 min and then stained for Smad2. The left panels show Smad2 staining, the middle panels show a merged image (Smad2: red; S100β-GFP/desmin: green; DAPI: blue), and the right panels show bright-field images. Diffuse cytoplasmic staining of Smad2 was seen in vehicle-treated GFP-positive cells (folliculostellate cells: Aa, Ab) and desmin-positive cells (pericytes: Ba, Bb); however, nuclear staining was seen in TGFβ2-treated GFP-positive cells (Ad, Ae) and desmin-positive cells (Bd, Be). The cells, Smad2 positive in the nucleus and desmin-negative, in Fig. Be are considered to be folliculostellate cell or endothelial cell, because they express TGFβ receptor II [28]. Arrowheads: nucleus of GFP-positive cells. Arrows: nucleus of desmin-positive cells. Bars=10 μm.

Fig. 3.

Relative mRNA expression level of fibromodulin in cell aggregates treated with TGFβ2 and/or selective TGFβ receptor I inhibitor (SB431542) evaluated by quantitative real-time PCR. Anterior pituitary cells of Wistar rats were used. mRNA expression levels were normalized with β-actin. A) Cell aggregates were treated with different concentrations of TGFβ2 (1–50 ng/ml). TGFβ2 induced fibromodulin synthesis in a dose-dependent manner (n=4, mean±SEM). B) Cell aggregates were treated with TGFβ2 (50 ng/ml) and SB431542 (10 μM). BSA and DMSO were the vehicle control for TGFβ2 and SB431542, respectively. SB431542 attenuated TGFβ2-induced fibromodulin synthesis (n=4, mean±SEM). *P<0.05 (Dunnett’s test).

Fig. 4.

Relative protein expression level of fibromodulin in cell aggregates treated with TGFβ2 evaluated by Western blotting. Anterior pituitary cells of Wistar rats were treated with TGFβ2 (50 ng/ml) for 5 days in hanging-drop culture. A) Top: Fibromodulin (Fmod); bottom: β-actin. B) The graph shows the results of immunoblot analysis as fold induction normalized against β-actin (n=3, mean±SEM). *P<0.05 (Student’s t-test).