Stimulation of sulfated‐proteoglycan synthesis by forskolin in monolayer cultures of rabbit articular chondrocytes

Abstract

Forskolin, a plant cardiotonic diterpene, stimulated proteoglycan biosynthesis by chondrocytes in monolayer culture. The quantitative increase in proteoglycans was dependent on the concentration of forskolin, but was relatively independent of the presence of serum. At forskolin concentrations that stimulated proteoglycan synthesis, a significant stimulation of adenylate cyclase and cAMP was also measured. The quantitative increase in proteoglycans was characterized, qualitatively, by an increased deposition of newly synthesized proteoglycan in the cell-associated fraction. An analysis of the most dense proteoglycans (fraction dA1) in the cell-associated fraction showed that more of the proteoglycans eluted in the void volume of a Sepharose CL-2B column, indicating that an increased amount of proteoglycan aggregate was synthesized in forskolin-treated cultures. The proteoglycan monomer dA1D1 secreted into the culture medium of forskolin-stimulated cultures overlapped in hydrodynamic size with that of control cultures, although cultures stimulated with forskolin and phosphodiesterase inhibitors produced even larger proteoglycans. The hydrodynamic size of 35SO4 and 3H-glucosamine-labelled glycosaminoglycans isolated from the dA1D1 fraction of the culture medium was greater in forskolin-treated chondrocytes, especially from those in which phosphodiesterase inhibitors had been added. These results indicated that forskolin, a direct activator of chondrocyte adenylate cyclase mimicked the effects of cAMP analogues on chondrocyte proteoglycan synthesis previously reported. These results implicate activation of adenylate cyclase as a regulatory event in the biosynthesis of cartilage proteoglycans, and more specifically in the production of hydrodynamically larger glycosaminoglycans.

Pre-transplantational Control of the Post-transplantational Fate of Human Pluripotent Stem Cell-Derived Cartilage

“…The PT7 condition showed the same tendency but with only a weak statistical significance. Thus, forskolin supports hyaline cartilage formation by suppressing COL1A1 expression and stimulating sGAG production from chondrocytes, consistent with a previous suggestion ( Malemud et al., 1986 ), regardless of the type of BMPs used.…”

IL-17 Inhibits Chondrogenic Differentiation of Human Mesenchymal Stem Cells

“…In agreement with these results, H89, a selective PKA inhibitor, strongly suppressed SOX9 phosphorylation ( Figure 3E ) and chondrogenesis ( Figure 5 ). These data implicate PKA in the phosphorylation of SOX9 and chondrogenic differentiation of human MSCs, which is consistent with previous reports indicating that PKA is a positive regulator of chondrogenic differentiation [38] , [39] . However, conflicting findings have been reported, demonstrating that H89 induced aggrecan, a chondrogenic marker [40] .…”

Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate aCol2a1 Chondrocyte-Specific Enhancer

“…In cultured chondrocytes, cyclic AMP (cAMP) enhances the expression of several markers of chondrocyte differentiation, such as Col2a1, link protein, and aggrecan (14,23,28); cAMP also mediates the effects of the parathyroid hormone-related peptide (PTHrP), a known modulator of chondrocyte differentiation in growth plates (12,29). Hence, we postulated that the interactions between SOX9 and PKA-C␣ may be physiologically relevant and asked whether SOX9 might be a target for PKA phosphorylation and whether such phosphorylation may affect the activity of SOX9.…”