Abstract
The alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA was examined in epiphyseal, costal, and meniscal cartilage from 3-week-old beagles and in nasal, tracheal, articular, and meniscal cartilage from 1- and 2-year-old Labrador retrievers. In contrast to the 100% expression of ED-B(+) mRNA that has been reported for embryonic chick cartilage (Bennett, V.D., Pallante, K.M., and Adams, S.K. (1991) J. Biol. Chem. 266, 5918-5924), all cartilages studied expressed both the ED-B(+) and ED-B(-) forms of fibronectin mRNA with the exception of the trachea, in which expression was 100% ED-B(-). Of all cartilages studied, only the meniscus had detectable levels of ED-A(+) mRNA. Placing articular cartilage chondrocytes in primary monolayer culture dramatically up-regulated the expression of ED-A(+) mRNA to 25% of the total, and this expression was further increased by the addition of transforming growth factor beta 1 or fucoidan to the culture medium. The expression of ED-B(+) mRNA remained at about 18% in the cultured chondrocytes and was not further affected by either transforming growth factor beta 1 or fucoidan. In contrast, dibutyryl cyclic adenosine monophosphate decreased the relative expression of both the ED-A(+) and ED-B(+) forms of fibronectin pre-mRNA. We concluded that the expression of ED-B(+) fibronectin remains relatively high in chondrocytes from cartilaginous canine tissues (15-35%) with the exception of the trachea, in contrast to the less than 10% expression of ED-B(+) fibronectin reported for other non-fetal tissues.
Highlights
Placing articular cartilage chondrocytes in primary monolayer culture dramatically up-regulated the expression of ED-A( +) mRNA to 25% of the total, and this expression was further increased by the addition of transforming growth factor In or fucoidan to the culture medium
In a previous preliminary study using the polymerase chain reaction,l we provided direct evidence that fibronectin mRNA is expressed in adult canine articular cartilage and that, in contrast to chick embryo cartilage, both the ED-B( +) and ED-B( -) forms of fibronectin are expressed (Zhang et al, 1993)
Canine Fibronectin ED-A and ED-B Sequences Are Highly Homologous to Other Mammalian Species-Canine fibronectin cDNAs were amplified successfully with the reverse transcription-PCR approach using cellular RNA from cultured chondrocytes as described under "Experimental Procedures." Both canine ED-A and ED-B fibronectin cDNAs were generated in this manner with primers derived from the human fibronectin sequence
Summary
Vol 270, No., Issue of January 27, pp. 1817-1822, 1995 Printed in U.S.A. Alternative Splicing of ED-A and ED-B Sequences of Fibronectin Pre-mRNA Differs in Chondrocytes from Different Cartilaginous Tissues and Can Be Modulated by Biological Factors*. In an attempt to understand the regulation of fibronectin synthesis and, more importantly, the alternative splicing of fibronectin pre-mRNA, a primary chondrocyte culture system was used to study the effects of transforming growth factor {31 (TGF-{311, dibutyryl cyclic adenosine monophosphate «Bu) cAMP), and fucoidan on fibronectin synthesis and the alternative splicing of the ED-A and ED-B segments of fibronectin pre-mRNA.
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