Limb development in chick embryos: Cyclic AMP‐dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis

Abstract

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20-23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25-28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP-dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80-85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane-enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP-dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP-protein phosphorylation system in the differentiation of limb tissues.

Protein Kinase A Regulates Chondrogenesis of Mesenchymal Cells at the Post-Precartilage Condensation Stage via Protein Kinase C-α Signaling

“…The fact that PKA regulates chondrogenesis of limb bud mesenchymal cells, the earliest step in limb bone development, has been known for almost two decades. Involvement of PKA has been indicated by the observed elevation of cAMP levels at the onset of differentiation (12,13) and by the presence of PKA activity throughout the differentiation period (14–16) . However, the mechanism by which PKA effects chondrogenesis is not yet clearly understood.…”

“…(12,13) In addition, treatment of the cells with reagents that elevate cellular cAMP levels also stimulates chondrogenesis of the tissue cultured cells. (2,13) Activity of PKA was detected at various stages of chondrogenesis, (14,15) and certain extracellular signaling molecules, such as bone morphogenic protein induced chondrogenesis by stimulating PKA. (16) However, the molecular mechanism of the cAMP/PKA regulation of chondrogenesis has not been studied thoroughly yet.…”

Influence of prostaglandins on DNA and matrix synthesis in growth plate chondrocytes

“…397 398 OKEEFE ET Al,.levels of prostaglandin E, precedes chondrogenesis, and PGE, has been further demonstrated to directly stimulate the chondrogenesis of undifferentiated limb mesodermal cells through a CAMP-mediated mechanism. (5)(6)(7)(8) Prior work demonstrated prostaglandin synthetase activity in homogenates of growth plate cartilage,'') and recently, prostaglandins of the E series were identified in normal growth plate cartilage in nanomolar concentrations. ( l o ) Prostaglandin E, was found to increase cartilaginous tissue mass in a fracture healing and prostaglandins El, E,, and D, were shown to stimulate the accumulation of cAMP in monolayer cultures of chondrocytes isolated from rabbit ribs.…”

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures

“…The inhibition of PP2A or/and PP1 by OA shifts the balance of reversible cellular protein phosphorylation toward increased phosphorylation of target proteins (Schönthal 1998). Some Ser/Thr specific protein kinases such as PKA (Smales and Biddulph 1985;Zhang et al 1996), PKC (Chang et al 1998) and receptor kinases of TGF-β-superfamily (Kulyk et al 1989;Leonard et al 1991) have been described as modulators of chondrogenesis. Signal transduction processes of PKA (Gonzalez and Montminy 1989), PKC (Xie and Rothstein 1995) and TGF-β pathways (Potchinsky et al 1997) can induce the phosphorylation of cyclic AMP response element binding protein (CREB) on Ser-133.…”