Focal Adhesion and Chondroinduction

Focal Adhesion is a statistically significant pathway involved in LSJL.  Whether LSJL stimulates or disrupts focal adhesion is unclear.

Cytoskeletal and Focal Adhesion Influences on Mesenchymal Stem Cell Shape, Mechanical Properties, and Differentiation Down Osteogenic, Adipogenic, and Chondrogenic Pathways.

"Osteogenic differentiation is more prevalent in MSCs with a stiff, spread actin cytoskeleton and greater numbers of focal adhesions. Both adipogenic differentiation and chondrogenic differentiation are encouraged when MSCs have a spherical morphology associated with a dispersed actin cytoskeleton with few focal adhesions."

"uniaxial[all along one axis like all along the lateral axis like with LSJL], unconfined compression and cyclic hydrostatic pressure increase chondrogenesis."

"If the perinuclear actin cap is inhibited, the formation of actin stress fibers in hMSC is prevented."

"chondrocytes have a stiffness that is midway between MSCs and adipocytes at 1.2 kPa."

"the actin cytoskeleton is not as important for mechanotransduction in chondrocytes as it is in osteoblasts."

" In chick wing-bud MSCs, disrupting the actin cytoskeleton with cytochalasin-D encouraged chondrogenesis. Various factors known to regulate the actin cytoskeleton play an important role in regulating chondrogenesis. As MSCs undergo chondrogenesis, they exhibit a decrease in RhoA activity. This decrease in RhoA is at least partially responsible for chondrogenic differentiation of MSCs, as MSCs made to overexpress RhoA exhibited decreased chondrogenesis. This shows that the cell causes the actin cytoskeleton to become more diffused through decreased RhoA activity to undergo chondrogenesis."

"Cytoskeletal configuration and regulation have also been shown to play an important role in chondrogenesis. Like adipogenesis, chondrogenesis is encouraged by having a rounded cell shape. MSCs plated on surfaces modified with RGD (arginine, glycine, aspartic acid) peptides spread out, whereas MSCs on RGE (arginine, glycine, glutamic acid) peptide-modified surfaces, which prevent focal adhesion attachment, remain rounded. MSCs on the RGD surfaces showed decreased chondrogenesis as evidenced by lower levels of mRNA for collagen II and aggrecan. These surfaces also exemplified the importance of interactions between integrins and the actin cytoskeleton in chondrogenesis. While cells seeded on RGD surfaces had high levels of localized vinculin expression, MSCs on RGE surfaces expressed only low levels of vinculin that were not localized. This implies that strong focal adhesion attachments are not necessary or beneficial to chondrogenesis."

"Active mechanical interventions can also cause actin–cytoskeletal alignment in MSCs. In 3D culture, MSCs show actin–cytoskeletal alignment parallel to the compression direction both in cyclic and static unconfined compression. Because the pellets are unconfined, there is likely an element of tensile strain in the direction perpendicular to applied compression. Therefore, it is not surprising that cyclic uniaxial stretch can also be used to cause actin cytoskeletal alignment in MSCs. We have shown that actin fiber alignment in the direction of applied strain occurs in hMSC exposed to uniaxial cyclic tensile strain."

Microgravity reduces RhoA organization and actin organization.  Chondrogenesis is encouraged by reduced spreading and a disrupted actin cytoskeleton.  DC electric fields also encourage a chondrogenic round cell shape.

"Oscillatory fluid flow, which has been shown to increase cytoskeletal organization, has also been reported to increase Sox9 production in MSCs when delivered at 1 Hz with a peak force of 1 Pa."<-so the correlation between cytoskeleton organization and chondroinduction is not perfect.

Less focal adhesions are more conductive to chondrogenesis.

MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells.

"we investigated the role of miRNA-488 in the cellular condensation, which is essential initiation for chondrogenic differentiation. We found that miRNA-488 expression is up-regulated at the precondensation stage and then down-regulated at the postcondensation stage. Blockade of miRNA-488 via the use of PNA (peanut agglutinin)-based ASOs (antisense oligonucleotides) decreased the protein level of integrins β1 and phosphorylated FAK (focal adhesion kinase) and resulted in the suppression of cell motility and migration. Moreover, in parallel with theses observation, treatment of anti-miRNA-488 oligonucleotides up-regulated the level of MMP (matrix metalloprotease)-2 activity, and co-treatment with GM6001, an MMP inhibitor, induced recovery of cellular condensation inhibited by blockade of miRNA-488. Collectively, our results suggest that miRNA-488 is one of regulator in cell to ECM (extracellular matrix) interaction through modulation of focal adhesion activity by MMP-2 during chondrogenesis of limb mesenchymal cells."

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells.

"Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling."

"The anti-proliferative effects of curcumin include blocks at specific cell cycle stages, particularly a G2/M block"

"Curcumin is known to inhibit a number of kinase and enzyme activities, including PKC, phosphorylase kinase, EGF receptor{up in LSJL} and erbB2"

"curcumin suppressed chondrogenesis by stimulating apoptotic cell death, down-regulating integrin β1{LSJL upregulates ITGB-Like 1, structurally it is similar to ITGB1 but functionally it is unclear} and lowering the phosphorylation level of FAK. This, in turn, lead to reorganization of the actin cytoskeleton via modulation of Akt signaling."

"members of the integrin family, including the α5β1 fibronectin receptor, α2β1 and α3β1, and the vitronectin receptor αvβ3 interact reversibly with both ECM proteins and cytoskeletal structures"

"Akt signaling positively regulates chondrogenic differentiation of chick limb bud mesenchymal cells via reorganization of the actin cytoskeleton to a cortical pattern with concomitant rounding of chondrogenic competent cells"<-LSJL increases Akt-phosphorylation.

"phosphorylation level of Akt was decreased when cells were exposed to curcumin"

" Undifferentiated chondrogenic progenitor cells are characterized by pronounced fibrillar actin fibers, whereas chondrocytes display a primarily cortical organization of their actin filaments "

Focal adhesion kinase/Src suppresses early chondrogenesis: central role of CCN2.

"FAK/Src signaling, which mediates cell/matrix attachment, suppresses early chondrogenesis, including the induction of Ccn2, Agc{up in LSJL}, and Sox6. The FAK/Src inhibitor PP2 elevates Ccn2, Agc, and Sox6 expression in wild-type mesenchymal cells in micromass culture, but not in cells lacking CCN2."

"The level and efficiency of overall chondrogenic differentiation, and hence subsequent bone development, is directly correlated to the density of the initial condensation"

" the formation of condensations and cartilage nodules requires adhesive signaling and remodeling of the actin cytoskeleton, via Rho and Rac"

WISP2, Cyr61, and Acan are upregulated in CCN2+/+ versus CCN2-/- as well as in LSJL.

"In the Fak-/- cells, L-Sox5 and Col2a1 mRNA expression was significantly higher than in the wild-type controls"

"CCN2 is a key modulator of adhesive signaling and promotes chondrogenic gene expression in vivo and in vitro"

"FAK signaling may suppress chondrogenesis by preventing the induction of CCN2."


Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment.

"Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology."

"Aggrecan binds specifically to hyaluronan via its N-terminal domain, thereby forming multimolecular aggregates consisting of 100 or more molecules. This creates a very high fixed charge density, allowing for water retention and resistance to deformation"

"Cells adhering via α2β1 to chondroadherin remain round, as do cells adhering via α5β1 to the integrin binding domain of fibronectin. In the case of fibronectin, treatment with a heparin-binding fragment of the molecule (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C is necessary to provide sufficient signals inducing spreading, formation of focal adhesions, and stress fibers. This process involves both integrin and syndecan receptors at the cell surface. Similarly, cell spreading can be induced on chondroadherin as a consequence of phorbol ester treatment"

"cell surface collagens mediate adhesion to matrilin-1. Binding of cells to chondroadherin was partially decreased upon collagenase treatment"

"anti-β1 integrin antibody prevented spreading, and the cells formed aggregates"

"the addition of EDTA, known to dissociate integrin interactions, lead to cell detachment"

"collagen type II (adhering to the α1β1, α2β1, α10β1, and α11β1 integrins), fibronectin (adhering primarily to the α5β1 integrin), laminin (adhering to the α6β1 integrin), vitronectin (adhering to the αVβ3 integrin), and chondroadherin (interacting only with the α2β1 integrin)."

"Adhesion of human chondrocytes to chondroadherin or peptide induces phosphorylation of ERK1/2."

MMP-2 functions as a negative regulator of chondrogenic cell condensation via down-regulation of the FAK-integrin beta1 interaction.

"we investigated the functional role of MMP-2 in chondrogenesis of leg bud mesenchymal cells. Small interference RNA (siRNA)-mediated knockdown of mmp-2{LSJL upregulates MMP2, maybe an MMP2 inhibitor would augment LSJL results} promoted precartilage condensation and chondrogenesis. Treatment with bafilomycin A1, an MMP-2 activator, or GM6001, an MMP inhibitor, at the pre-condensation stage resulted in the inhibition or promotion of chondrogenesis, respectively. By comparison, treatment at the post-condensation stage had little or no effect on chondrogenesis. These results indicate that MMP-2 is involved in the regulation of cell condensation. Inhibition of MMP-2 activity by mmp-2 specific siRNA increased the protein level of fibronectin, and integrins alpha5 and beta1. The interaction between focal adhesion kinase (FAK) and integrin beta1 leading to tyrosine phosphorylation of FAK was also enhanced. Moreover, inactivation of p38MAPK down-regulated the level of MMP-2 mRNA and activity, and increased mesenchymal cell condensation in parallel with enhanced phosphorylation of FAK. Taken together, our data indicate that MMP-2 mediates the inhibitory signals of p38MAPK during mesenchymal cell condensation by functioning as a negative regulator of focal adhesion activity regulated by FAK via interactions with fibronectin through integrin beta1."

"testican-1, an inhibitor of pro-MMP-2 activation, is expressed in joint and growth plate cartilage"

"the activity of MT1–MMP on the cell surface is constitutively down-regulated through a vacuolar H+-ATPase-dependent degradation process."

"Blockade of this degradation caused the accumulation of TIMP-free active MT1–MMP molecules on the cell surface and pro-MMP-2 activation was strongly enhanced as a consequence of this impaired degradation."

"FAK is localized to focal adhesions where it is activated by phosphorylation after integrin-mediated cell attachment"

"The tyrosine phosphorylation of FAK increased after knockdown of mmp-2, but decreased after activation of MMP-2"

"Adhesion of chondrocytes to the site of interaction between fibronectin and integrin β1 leads to tyrosine phosphorylation of cytoskeletal and signaling proteins localized at focal adhesions, including paxillin, c-Src and FAK"

"MMP-2 inhibits cellular condensation by down-regulating the integrin β1-mediated interaction between fibronectin and FAK, and tyrosine phosphorylation of focal adhesion components, such as FAK, paxillin and c-Src at the focal adhesion site. The p38MAPK pathway induces the down-regulation of tyrosine phosphorylation of FAK, via the up-regulation of MMP-2, at the transcription and protein activity levels, leading to inhibition of leg bud mesenchymal cell condensation."