The MEK-ERK Signaling Pathway Is a Negative Regulator of Cartilage-specific Gene Expression in Embryonic Limb Mesenchyme

Abstract

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK kinase cascade, has recently been implicated in the regulation of embryonic cartilage differentiation. However, its precise role in this complex process remains controversial. To more thoroughly examine the role of the MEK-ERK kinase cascade in chondrogenesis, we analyzed the effects of two structurally different pharmacological inhibitors of MEK, the upstream kinase activator of ERK, on chondrocyte differentiation in micromass cultures of embryonic chick limb mesenchyme cells. We found that the MEK inhibitors, U0126 and PD98059, promote increased accumulation of cartilage-characteristic mRNA transcripts for type II collagen, aggrecan, and the transcription factor, Sox9. PD98059 treatment stimulated increased deposition of sulfated glycosaminoglycan into both Alcian blue-stainable cartilage matrix and the surrounding culture medium, whereas U0126 elevated glycosaminoglycan secretion into the medium fraction alone. Both MEK inhibitors increased total type II collagen protein accumulation in micromass culture and elevated the activity of a transfected type II collagen enhancer-luciferase reporter gene. Thus, pharmacological MEK inhibition induced increased expression of multiple chondrocyte differentiation markers. Conversely, transfection of limb mesenchyme cells with a constitutively active MEK1 plasmid resulted in a prominent decrease in the activity of a co-transfected type II collagen enhancer-luciferase reporter gene. Collectively, these findings support the hypothesis that signaling through the MEK-ERK kinase cascade may function as an important inhibitory regulator of embryonic cartilage differentiation.

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

“…These findings are consistent with previous studies that demonstrated inhibition of aggrecan expression by H 2 O 2 in juvenile bovine chondrocytes (57) and negative regulation of aggrecan expression by ERK in embryonic chick limb mesenchyme (50). The ERK inhibition of aggrecan is probably through the activation of its downstream target, c-Fos, which has been found to directly reduce aggrecan expression in the chondrosarcoma cell line HCS 2/8 cells (58).…”

“…Infection of chondrocytes with lentivirus expressing CA MEK reduced both aggrecan mRNA expression and proteoglycan protein synthesis, as did activation of MEK-ERK using tBHP. Negative regulation of proteoglycan production by activated MEK-ERK has been previously observed during chondrogenesis in embryonic chick limb bud cultures (50), and inhibition of MEK-ERK has been found to promote chondrocytic differentiation of ATDC5 cells (51) and aggrecan expression in immortalized rat chondrocytes (52). These findings are in contrast to a study of mechanically stimulated bovine calf chondrocytes, where inhibition of MEK-ERK either reduced aggrecan expression or had no effect, depending on the stimulation conditions (53).…”

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK

“…Also, similar to our findings, inhibition of ERK activation with PD98059 did not block IGF-I-stimulated proteoglycan synthesis by mesenchymal cells, but instead promoted it [26]. MEK inhibition was also found to increase the expression of chondrocyte matrix genes by chick limb mesenchyme [27]. In alginate cultures, we noted an increase in proteoglycan synthesis when cells were treated with IGF-I after MEK inhibition with PD98059 or U0126.…”

PTH Stimulated Growth and Decreased Col‐X Deposition are Phosphotidylinositol‐3,4,5 Triphosphate Kinase and Mitogen Activating Protein Kinase Dependent in Avian Sterna

“…As discussed in the introduction, 10 lM for the PI3K inhibitor had worked well in this lab in previous investigations and delayed chondrocyte differentiation in micromass cultures and mouse tibiae (Ulici et al, 2008). The range of 10-100 lM for the MAPK inhibitor was derived from chondrocyte, cartilage, bone or PTH/PTHrP studies, which had previously used PD98059 (Beier et al, 1999;Bobick and Kulyk, 2004). The majority of these studies had used 20 lM in cell culture.…”