We have shown that dynamic mechanical signals vitally control AC proliferation and differentiation by regulating the MAPK signaling cascade. Furthermore, the actions of mechanical signals are sustained in the presence of proinflammatory signals induced by IL-1β. We have exposed ACs to dynamic tensile forces to assess their potential in controlling cell growth. During joint movement, ACs simultaneously experience dynamic compression-, tension-, and torsion-induced forces. In vitro
, ACs subjected to 10% compression in three-dimensional microfiber or agarose constructs exhibit many biochemical changes similar to those of ACs exposed to 6% tensile forces. For example, 10% compressive forces as well as 6% tensile forces suppress proinflammatory gene induction, upregulate total proteoglycan contents, and aggrecan, collagen type II, and SOX-9 mRNA induction in ACs [7
]. Therefore, in this study, 6% tensile forces were used to examine the signaling events induced by DS. However, so far, the extent of compressive or tensile forces experienced by ACs during joint movement in vivo
is not clear.
Intracellular signal transduction by mechanical signals begins with ILK activation. This was evident by the observations that mechanical signals failed to induce ERK1/2 phosphorylation in ACs transfected with mutant-ILK or kinase activity-deficient ILK plasmids. However, mechanical signals induced ERK1/2 activation in ACs transfected with WT ILK or untransfected cells. These studies revealed that ILK activation by mechanical signals is of critical importance given the fact that integrins are the putative mechanosensors of chondrocytes, and ILK is one of the central signaling components of the integrin complex [15
]. Interestingly, mechanical signals are also perceived via integrins to activate Rho GTPases to regulate cytoskeletal rearrangements [33
]. This indicates that mechanical signals regulate diverse cellular functions via integrin engagement.
Mechanoactivation of ACs leads to the rapid activation of RAS. In an effort to examine whether mechanical signals regulate RAS during inflammation, we examined the effects of IL-1β on RAS activation. IL-1β induces minimal activation of RAS. Nevertheless, RAS activation is similar in mechanoactivated cells irrespectively of the presence of IL-1β. RAS activation is associated with ERK1/2-mediated cell proliferation [34
]. Consistent with these findings, our data show that the RAS inhibitor GGT12133 attenuates ERK1/2 phosphorylation induced by mechanical signals. RAS activation is central to activation of many cell surface receptors, such as growth factor receptors, receptor tyrosine kinases, integrins, and IL-6 receptors [34
], further suggesting that dynamic mechanical signals activate signaling molecules similar to other growth factors.
To examine how mechanical signals and IL-1β regulate ERK1/2 signaling cascade that result in differential gene expression, we next examined the activation of Rafs [37
]. Mechanical signals trigger c-Raf kinase activity by phosphorylating Ser338 residues. However, IL-1β induces Ser445-B-Raf phosphorylation. B-Raf was not activated by mechanical signals. However, mechanical signals inhibited IL-1β-induced B-Raf activation. This disparity in the activation of Rafs may play a critical role in the differential processing of signals generated by IL-1β and mechanical forces. However, the mechanisms that underlie this regulation of c-Raf and B-Raf remain to be elucidated.
Activation of B-Raf by IL-1β or c-Raf by mechanical signals results in MEK1/2 activation via Ser217/221 phosphorylation [19
]. Subsequently, MEK1/2 activates ERK1/2 by phosphorylating both Thr202/Tyr204 residues. Following mechanoactivation, phosphorylated ERK1/2 rapidly translocates to the nucleus and is redistributed to the cell surface. ERK proteins after activation translocate to the nuclear compartment, where they act as the main executor of ERK1/2 biological functions, and channel a diverse array of signals via downstream targets. Additionally, ERK dimers and scaffolds translocate to cognate cytoplasmic substrates, where they stabilize ERK1/2 and Myc functions in cell proliferation [35
Interestingly, ERK1/2 activation is temporally regulated in response to DS as well as IL-1β. DS rapidly induces ERK1/2 phosphorylation, which is observed within 10 minutes. IL-1β-induced ERK1/2 phosphorylation is apparent at 30 minutes. It is likely that DS, by activating kinases upstream of ERK1/2, initiates a feedback loop that suppresses IL-1β-induced ERK1/2 activation. Such early activation of ERK1/2 by DS may likely play a role in sustaining its effects in the presence of IL-1β.
Mechanoactivation of ACs leads to c-Myc, VEGF, and SOX-9 mRNA expressions, all of which have been implicated in the proliferative response of cells to a variety of stimuli [35
]. Furthermore, ERK1/2 activation is required for c-Myc, SOX-9, and VEGF mRNA expression, as evidenced by the suppression of their transcriptional activation by PD98059. We have also observed that ERK1/2 activation by IL-1β fails to induce SOX-9 or VEGF expression. This may explain the suppression of AC proliferation in the presence IL-1β. These findings again point to similarities between mechanical signals and other growth factors that use the ERK1/2/Myc signaling cascade to regulate cell proliferation [23
]. Furthermore, the fact that mechanical signals upregulate c-Myc, SOX-9, and VEGF in the presence of IL-1β supports the benefits of mechanoactivation of ACs in the inflamed cartilage.