Elsevier

Bone

Volume 33, Issue 6, December 2003, Pages 911-918
Bone

Original article
Connective tissue growth factor mRNA expression pattern in cartilages is associated with their type I collagen expression

https://doi.org/10.1016/j.bone.2003.07.010Get rights and content

Abstract

Connective tissue growth factor (CTGF) has been identified as a secretory protein encoded by an immediate early gene and is a member of the CCN family. In vitro CTGF directly regulates the proliferation and differentiation of chondrocytes; however, a previous study showed that it was localized only in the hypertrophic chondrocytes in the costal cartilages of E 18 mouse embryos. We described the expression of CTGF mRNA and protein in chondrocytes of different types of cartilages, including femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and cartilage formed during the healing of mandibular ramus fractures revealed by in situ hybridization and immunohistochemistry. To characterize the CTGF-expressing cells, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression. Among these different types of cartilages we found distinct patterns of CTGF mRNA and protein expression. Growth plate cartilage and the costal cartilage showed localization of CTGF mRNA and protein in the hypertrophic chondrocytes that expressed type X collagen mRNA with less expression in proliferating chondrocytes that expressed type II collagen mRNA, whereas it was also expressed in the proliferating chondrocytes that expressed type I collagen mRNA in the condylar cartilage, the articular cartilage, and the cartilage appearing during fracture healing. In contrast, the growth plate cartilages or the costal cartilages were negative for type I collagen and showed sparse expression of CTGF mRNA in the proliferating chondrocytes. We found for the first time that CTGF mRNA could be differentially expressed in five different types of cartilage associated with those expressing type I collagen. Moreover, the spatial distribution of CTGF mRNA in the cartilages with type I collagen mRNA suggested its roles in the early differentiation, as well as in the proliferation and the terminal differentiation, of those cartilages.

Introduction

Connective tissue growth factor (CTGF) belongs to the CCN family (Cyr61/Cef10, CTGF/Fisp-12, Nov) [1], which comprises secretory proteins that contain 38 conserved cysteine residues with four specific domains. It was first identified as a mitogenic factor produced by human umbilical vein endothelial cells [2]. It was also obtained as a chondrocyte or chondrosarcoma cell line (HCS)-specific DNA fragment by differential display PCR and was expressed selectively in the hypertrophic region of costal cartilage and in the cartilage tissues in the embryonic vertebral column [3] In vitro, CTGF promotes the proliferation and differentiation of chondrocytes [4]; through a p44/42 MAPK/extracellular-signal-regulated kinase and through a p38 mitogen-activated protein kinase, respectively [5]. In vivo, CTGF mRNA is localized in the hypertrophic chondrocytes, but not in the proliferating chondrocytes in the embryonic costal cartilages of the mouse [3]. This distribution pattern suggests that CTGF might play some roles in terminal differentiation of the chondrocytes and in angiogenesis during the process of endochondral ossification. In addition, it is also detected in the proliferating chondrocytes during fracture healing in mouse ribs, suggesting putative roles in chondrocyte proliferation in fracture healing [6].

There are several functions of cartilages. The growth plate cartilage represents regions of growth, and bone elongation occurs by proliferation of chondrocytes and subsequent endochondral ossification. In contrast, the articular cartilage covers the surfaces of the moving joints. The mandibular condylar cartilage is known to function as an articular cartilage and also as growth cartilage and is loaded during physiological jaw movement such as mastication. On the other hand, cartilages also appear ectopically during the process of bone fracture healing. It is a complex process that involves an immediate response to injury, chondrogenesis, and endochondral ossification [7], [8], and the edges of the fractured bone are also subjected to mechanical stress in this process.

In the present study, we investigated the possible differences in the distribution pattern of CTGF in five different types of cartilages, i.e., femoral growth plate cartilage, costal cartilage, femoral articular cartilage, mandibular condylar cartilage, and the cartilage that appears during fracture healing. To characterize the CTGF mRNA-positive chondrocytes, we also analyzed the distribution of the type I, type II, and type X collagen mRNA expression.

Section snippets

Tissue preparation and histology

Six-week-old male Wistar rats weighing 160–180 g were used. The experiment was approved by the animal committee of our school. Under anesthesia the animals were perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, and then their femurs, ribs, and mandibles were dissected. For mandibular ramus fracture, animals were anesthetized with pentobarbital sodium (40 mg/kg). An incision was made anteroinferior to the periosteum on the right side of the mandible in a sterile field. The

Results

Fig. 1 shows the histology of a saggital section of the femoral growth plate cartilage (Fig. 1A), of articular cartilage (Fig. 1A), and of mandibular condylar cartilage (Fig. 1B).

Fig. 2 shows the temporal histological changes during fracture healing of the mandibular ramus 0 (D), 3 (A), 7 (B), 14 (C), and 28 (E) day after fracturing. By day 3 after the bone had been fractured, undifferentiated mesenchymal cells enveloped the bone surface around the fracture site (Fig. 2A). By day 7, in the

Discussion

The present study showed the spatial distribution of CTGF mRNA in the femoral growth plate cartilage, femoral articular cartilage, and the mandibular condylar cartilage, each of which showed well-aligned chondrocyte layers. We also evaluated the temporal and spatial distribution patterns of CTGF mRNA expression in the process of the repair of the fractured mandibular ramus, where the cartilage appears transiently in time- and site- specific manners. Our findings revealed a differential

Acknowledgements

This study was supported by grants-in-aid (13667183, 13672152) for scientific research from the Ministry of Education, Science, and Culture of Japan. We thank Drs. Tohru Nakanishi, Satoshi Kubota, and Takashi Nishida for useful discussions.

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