Transient anabolic effects accompany epidermal growth factor receptor signal activation in articular cartilage in vivo

INTRODUCTION: Signals from the epidermal growth factor receptor (EGFR) have typically been considered to provide catabolic activities in articular cartilage, and accordingly have been suggested to have a causal role in osteoarthritis progression. The aim of this study was to determine in vivo roles for endogenous EGFR signal activation in articular cartilage. METHODS: Transgenic mice with conditional, limb-targeted deletion of the endogenous intracellular EGFR inhibitor Mig-6 were generated using CreLoxP (Mig-6-flox; Prx1Cre) recombination. Histology, histochemical staining and immunohistochemistry were used to confirm activation of EGFR signaling in the articular cartilage and joints, and to analyze phenotypic consequences of Mig-6 loss on articular cartilage morphology, proliferation, expression of progenitor cell markers, presence of chondrocyte hypertrophy and degradation of articular cartilage matrix. RESULTS: The articular cartilage of Mig-6-conditional knockout (Mig-6-cko) mice was dramatically and significantly thicker than normal articular cartilage at 6 and 12 weeks of age. Mig-6-cko articular cartilage contained a population of chondrocytes in which EGFR signaling was activated, and which were three to four times more proliferative than normal Mig-6-flox articular chondrocytes. These cells expressed high levels of the master chondrogenic regulatory factor Sox9, as well as high levels of putative progenitor cell markers including superficial zone protein (SZP), growth and differentiation factor-5 (GDF-5) and Notch1. Expression levels were also high for activated β-catenin and the transforming growth factor beta (TGF-β) mediators phospho-Smad2/3 (pSmad2/3). Anabolic effects of EGFR activation in articular cartilage were followed by catabolic events, including matrix degradation, as determined by accumulation of aggrecan cleavage fragments, and onset of hypertrophy as determined by type × collagen expression. By 16 weeks of age, the articular cartilage of Mig-6-cko knees was no longer thickened and was degenerating. CONCLUSIONS: These results demonstrate unexpected anabolic effects of EGFR signal activation in articular cartilage, and suggest the hypothesis that these effects may promote the expansion and/or activity of an endogenous EGFR-responsive cell population within the articular cartilage

Roles of Transforming Growth Factor-α and Epidermal Growth Factor in Chick Limb Development

AbstractWe have examined the distribution of transforming growth factor-α (TGF-α), epidermal growth factor (EGF), and the chicken EGF receptor (c-erbB), in embryonic chick limbs. Prior to limb budding, TGF-α is present in prospective limb-forming mesoderm and in prospective apical ectodermal ridge (AER)-forming ectoderm, but is not detected in non-limb-forming flank mesoderm or ectoderm, nor in presumptive non-AER-forming limb ectoderm, suggesting possible roles in initial limb formation and AER induction. Consistent with this possibility, TGF-α is present in the mesoderm of the wing buds of the amelic chick mutantslimblessandwingless, which form and bud normally, but is absent fromlimblessandwinglessectoderm, which fails to form an AER. TGF-α and EGF are present in the AER of the developing limb, and TGF-α, EGF, and c-erbBare present in the underlying subridge mesoderm, suggesting possible roles in reciprocal AER/subridge mesoderm interactions required for limb outgrowth. We found that exogenous TGF-α and EGF can promote the outgrowth of limb mesoderm in the absence of the AERin vitroand can also promote the outgrowth oflimblessandwinglesswing bud explants. EGF is present in ventral but not dorsal limb ectoderm, suggesting a role for EGF in specification of ventral ectoderm. TGF-α and EGF are not detected in the differentiating cartilaginous elements or muscle primordia of the limb, suggesting that cessation of TGF-α and EGF expression may be required for cartilage and muscle formation. We have found that exogenous TGF-α and EGF inhibit chondrogenesis and myogenesis of limb mesenchymein vitro.Together these results indicate that signaling through the EGF receptor via endogenous TGF-α and EGF may be important for initial limb formation, AER induction, outgrowth of limb mesoderm, and regulation of limb chondrogenic and myogenic differentiation

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb