Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

Abstract

During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation, whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development.

Figure 1. Pull down assay of Rho GTPase activity in immature and mature chondrocytes

Immature (LS) and mature (US) chondrocytes were isolated from caudal and cephalic regions of sterna from 17-day old chick embryos, respectively, and cultured until confluent. Whole cell lysates from LS and US cultures were prepared and subjected to a pull down assay as described in Materials and Methods. A, The contents of active forms of Rho GTPases (Active) that bound to Rhotekine binding domain for RhoA or Pak-1 binding domain for Rac-1 and Cdc42 were visualized by immunoblotting with anti-RhoA antibody (Rho), anti-Rac-1 antibody (Rac) or anti-Cdc42 (Cdc). The total cell lysates were also subjected to immunoblotting for RhoA (Total, lanes 1 and 2), Rac-1 (Total, lanes 3 and 4), Cdc42 (Total, lanes 5 and 6) or tubulin (Tubulin, lanes 1–6) as an internal loading control. B, Band intensities for active forms of the Rho GTPases were measured by ImageJ and represented as the relative ratio to the LS culture after normalization by the tubulin band intensity.

Figure 2. Effects of Rho GTPase activation on chondrocyte morphology

LS chondrocytes were isolated and infected with RCAS virus encoding CA-RhoA (C and G), CA-Rac-1 (D and H) or CA-Cdc42 (E and I) or insert-less virus (Control, B and F), and re-plated. A, The whole cell lysates were then prepared and subjected to immunoblot for tubulin (Tubulin) or myc (Myc) that had been tagged to CA-GTPases. B-I, The phase contrast pictures were taken 2 days (B-E) and 5 days (F-I) after passage. Arrows indicate large, spread cells in CA-Rac-1 expressing cultures. Scale bar represents 100 μm.

Figure 3. Expression of CA- or DN-Rac-1 proteins and Rac-1 activity in CA- or DN-Rac-1 RCAS virus infected chondrocytes

LS chondrocytes were isolated and infected with RCAS virus encoding CA- or DN-Rac-1 or insert-less virus (Cont). On day 7, whole cell lysates of these cultures were prepared and subjected to a pull down assay to examine Rac-1 activity. A, The proteins bound to Pak-1 beads were analyzed by immunoblot for Rac-1 (Active). The total cell lysates were subjected to immunoblot with the antibodies against Rac-1 (Total), myc (Myc) and tubulin (Tubulin). The arrow indicates the endogenous protein, while the arrowhead is the exogenous protein. B, The band intensity of active Rac-1 was measured by ImageJ and represented as a relative ratio to control after normalization with tubulin.

Figure 4. Effects of CA- and DN-Rac-1 expression on the cell attachment and spreading of chondrocytes

The LS cells were infected with RCAS virus encoding CA- (closed column) or DN-Rac-1 (shaded column) or insert-less control RCAS virus (Control, open column) and then re-plated on 24-well plates that had been coated with collagen type I (Collagen I), fibronectin, gelatin or collagen type II (Collagen II) in serum-free medium. The cultures were fixed with 10% formalin 1 hour (A) or 3 hours (B) after plating and stained with crystal violet. The number of cells attached (A) and the spreading areas of cells (B) in ten fields were counted as described in Materials and Methods. (* is p < 0.05, ** is p < 0.01 and *** is p < 0.005)

Figure 5. Effects of CA and DN-Rac-1 expression on chondrocyte function

The LS cells were infected with RCAS virus encoding CA- or DN-Rac-1, or insert-less RCAS virus (Control). The cultures were re-plated on collagen type I coated 24 well plates, maintained for 7 days and stained with Alcian blue (A–C) or for alkaline phosphatase (ALPase) (D–F). G and H, Total RNA were prepared from the control (open column), CA- (closed column) or DN-Rac-1 (shaded column) cultures on day 5 after re-plating and subjected to semiquantitative PCR to analyze the expression of Sox9, aggrecan (Agg), collagen type IX (Col IX) and MMP-7, -9 and -13.

Figure 6. Distribution of GFP-labeled Rho GTPases in chondrocytes

The LS cells were infected with RCAS virus encoding GFP-Rac-1 (A and B), GFP-RhoA (C), GFP-Cdc42 (D), Actin-GFP (E), or GFP (F). The pictures were taken under the fluorescence microscope on day 5. B, Phase contrast picture of the same field in A. Expression of these proteins did not induce significant alterations in chondrocyte function (data not shown). Note that GFP-Rac-1 and Actin-GFP were distributed at the cell membrane (arrows) while GFP-Cdc42 and GFP-RhoA were diffusely localized in cytoplasmic and GFP is found in both cytoplasm and nuclei. Scale bar represents 10 μm.

Figure 7. Comparison of GFP-Rho GTPase localization with F-actin

The LS cells were infected with RCAS virus encoding GFP-RhoA (A–C), GFP-Rac-1 (D–F), or GFP-Cdc42 (G–I), re-plated onto collagen type I coated coverslips and fixed with 10% formalin 1 hour after plating. The cells were stained with rhodamine-labeled phalloidin (B, E, and H). A, D and G were GFP images of the same fields of B, E and H, respectively. A merged picture demonstrated the co-localization of actin and GFP-Rac-1 (F), which is not seen in under GFP-RhoA or –Cdc42 (C and I). Scale bar represents 10 μm.

Figure 8. Changes in distribution of GFP-Rac-1 induced by ascorbic acid or Wnt 3A

The LS cells were infected with RCAS virus encoding GFP-Rac-1 or Actin-GFP and treated with 10 μg/mL of ascorbic acid (C and D, VC) or 30% of Wnt 3A conditioned medium (E and F, Wnt3A). Two days after treatment, GFP distribution was observed under a fluorescence microscope. Scale bar represents 10 μm.

Figure 9. Functional interaction between Wnt3A and Rac-1

LS cells were treated with 30% of Wnt3A or control conditioned media (CM) for two days. A, RNA was isolated from treated cells and subjected to RT-PCR for Tiam1 and HPRT expression. B, Lysates were also isolated from treated cells subjected to a biochemical pull down assay for active Rac-1 protein (Active). Total lysates were used to immunoblot for total Rac-1 protein (Total) or tubulin as a loading control (Tubulin). C, Densitometric analysis was performed for band intensities using ImageJ. D, LS cells were infected with control or DN-Rac-1 virus and treated with 5% of control (open column) or Wnt3A (closed column) containing conditioned media for one day. Cultures were harvested and subjected to the attachment assay described in Materials and Methods. (* is p < 0.05)