Comparison of cyclic compressive loading gene expression to LSJL gene expression

Here's the genes expressed during LSJL.  Not by chondrocytes but in adult bone marrow there are no chondrocytes to express the chondrogenic genes.

Note this study had successful chondroinduction so if the genes are similar that bodes well for LSJL.

Temporal expression patterns and corresponding protein inductions of early responsive genes in rabbit bone marrow-derived mesenchymal stem cells under cyclic compressive loading.

"Cyclic compressive loading may promote chondrogenesis of rabbit bone-marrow mesenchymal stem cells (BM-MSCs) in agarose cultures through the transforming growth factor (TGF)-beta signaling pathway. The activating protein 1 (AP-1) (Jun-Fos) complex mediated autoinduction of TGF-beta1 and its binding activity [are] essential for promoting chondrogenesis of mesenchymal cells[overexpression of c-Fos has been shown to inhibit chondrogenesis, maybe it depends on the cell line ATDC5 cells are already pro-chondrogenic and c-Fos may inhibit chondrogenesis in this cell type but not other cell types, LSJL has never used this cell line], whereas Sox9 was identified as an essential transcription factor for chondrogenesis of embryonic mesenchymal cells. The objective of this study was to examine temporal expression patterns of early responsive genes (Sox9, c-Fos, c-Jun, and TGF-beta type I and II receptors)[LSJL increases c-Fos and TGF-beta RI) and induction of their corresponding proteins in agarose culture of rabbit BM-MSCs subjected to cyclic compressive loading. The rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell-agarose constructs were made by suspending BM-MSCs in 2% agarose gel (10(7) cells/ml) for cyclic, unconfined compression tests performed in a custom-made bioreactor. In the loading experiment, specimens were subjected to sinusoidal loading with a magnitude of 15% strain at a frequency of 1 hertz for 4 hours per day. Experiments were conducted for 2 consecutive days. This study showed that cyclic compressive loading promoted gene expressions of Sox9, c-Jun, and both TGF-beta receptors and productions of their corresponding proteins, whereas those gene expressions exhibited different temporal expression patterns among genes and between 2 days of testing. The gene expression of c-Fos was detected only in the samples subjected to 1-hour dynamic compressive loading.  The TGF-beta signal transduction and activities of AP-1 and Sox9 are involved in the early stage of BM-MSC chondrogenesis promoted by dynamic compressive loading."

"specimens of the loading group were placed into the testing chamber of the bioreactor developed previously  and then preloaded with 5% static strain for 300 seconds. Following preloading, sinusoidal compressive loading was applied with a magnitude of 15% at a frequency of 1 hertz for 4 hours. After the unconfined compression test, specimens were unloaded and incubated for 20 hours to allow constructs to recover. Experiments were conducted for 2 consecutive days. Specimens of the control group were cultured in serum-free medium that was also used in the compression tests. The culture medium was changed every day after the compression test. For each day of testing, gene expressions of the loading group were analyzed at three time points (after 1, 2, and 4 hours of loading) during the 4-hour compression test and three time points (after 4, 8, and 20 hours of rest) during the recovery period. To examine protein induction, the samples, which were subjected to 2 and 4 hours of loading and recovered for 2 and 4 hours after 4-hour loading at the first day of testing, were used for protein analyses. Samples of the control group were analyzed at the beginning of the compression test and the recovery period."

"The intracellular TGF-β signal transduction is initiated by type I receptor (TβR-I) after its phosphorylation by ligand-bound type II receptor (TβR-II)"

"Activating protein 1 (AP-1), a transcription factor complex of the Jun and Fos nuclear oncoproteins, is one of the downstream targets for mitogen-activated protein kinase (MAPK) signaling pathway. MAPKs regulated chondrogenesis of chick embryo limb bud cells at postprecartilage condensation stages"

"overexpression of c-Fos gene inhibited chondrocyte differentiation of ATDC5 chondrogenic cells"

"During the 4-hour compression test, gene expressions of c-Jun, Sox9, type II collagen, TGF-β1, TβR-I, and TβR-II for the loading group gradually increased and reached the peak after 2 hours of loading and then decreased from that peak after 4 hours of loading. Only the loading group exhibited a weak expression of c-Fos gene after 1 hour of loading . After the 4-hour compression test, all gene expressions of the loading group decreased to a level similar to (i.e., c-Jun, type II collagen, TGF-β1, and TβR-II) or slightly higher than (i.e., Sox9) those of the control group except that the expression of the TβR-I gene exhibited another peak after 8 hours of rest "

"the significant upregulation of TβR-I and TβR-II gene expressions occurred earlier than that of type II collagen gene expression during the compression test"<-which may explain why type II collagen wasn't observed in the LSJL gene expression study.

"c-Jun and c-Fos proteins can form a stable heterodimer (AP-1 complex) with a high affinity for the DNA target sequence. It has been shown that the AP-1 complex can positively regulate c-Jun, whereas the binding of AP-1 complex to the promoter regions of TGF-β1 gene mediated autoinduction of TGF-β1. This study found that the induction of c-Fos gene expression was detected earlier than the significant upregulation of c-Jun and TGF-β1 gene expressions during the dynamic compression test. It may suggest that AP-1 complex was formed by dimerizing the newly synthesized c-Fos protein with the pre-existing c-Jun protein and then upregulated the expression of c-Jun and TGF-β1 genes[so maybe if LSJL doesn't upregulate c-Fos it doesn't matter?]. TGF-β1 treatment [is] able to activate gene expressions of c-Jun and c-Fos, TGF-β1 upregulation by dynamic compression loading may be able to mediate transcription of c-Fos and c-Jun through feedback mechanism. Activity of the AP-1 complex may play an important role in regulating chondrocyte differentiation of chondrogenic cell lines and limb mesenchymal cells as well as TGF-β–induced type II collagen expression in chondrocytes. Dynamic compressive loading may promote chondrogenic gene expressions of BM-MSCs through activation of the AP-1 complex."

Development of methods for studying the differentiation of human mesenchymal stem cells under cyclic compressive strain.

"hMSC were successfully isolated, purified using D7-FIB antibody, cloned, and characterized. The cells were subsequently analyzed using fluorescence-activated cell sorting using a panel of antibodies and differentiation into multiple cell lineages. D7FIB-positive cells were then seeded into collagen-alginate scaffolds and subjected to 10% or 15% cyclic compressive strain for 4 out of 24 hours for up to 21 days in a bespoke servo-assisted displacement-controlled device. Cells were analyzed using adenosine triphosphate assay to determine cell number, live-dead cell assay, and quantitative real-time polymerase chain reaction at 7 and 21 days. Cloned D7-FIB-positive hMSCs showed evidence of differentiation to an osteogenic lineage under 10% cyclic compressive strain alone (core binding factor alpha 1 (CBFA-1) was significantly upregulated at 7 and 21 days by a factor of 18.3 and 32.2, respectively) and to an osteo-chondrogenic lineage under 15% cyclic compressive strain alone (increased expression of CBFA-1, Sox9, and aggrecan). A combination of a composite viscoelastic scaffold and controlled cyclic compressive strain may be useful for study of the differentiation of MSC."

So heavier loads may be more effective at inducing chondrogenesis.

"Cell-seeded scaffolds were then subjected to cyclic compressive strain of 10% or 15% (in separate experiments) using a sinusoidal waveform with a frequency of 1Hz for 4 hours per day for 21 days. The strain amplitude was controlled to within 0.5% across all stations, and the 0 position was controlled to within 1% across all wells. After 7 and 21 days of culture, six statically cultured and six dynamically cultured scaffolds were harvested"

"Sox9 was significantly upregulated at 7 and 21 days by factors of 1.3 and 1.6, respectively, under static culture. Under dynamic conditions, Sox9 at 7 and 21 days was significantly upregulated by factors of 2.6 and 1.9, respectively. Aggrecan was significantly upregulated at 7 and 21 days by factors of 1.4 and 1.6, respectively, under static culture. Under dynamic conditions, aggrecan was significantly upregulated at 7 and 21 days by factors of 1.3 and 2.0, respectively."<-LJSL gene expression was done after 49 hours so it may not have been long enough to induce Aggrecan and Sox9 expression.

So maybe the loads weren't heavy enough to induce chondrogenic gene expression and an increase in load by 50% so 0.75N for rats may be more chondrogenic.

Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.

"Unconfined cyclic compression [can] induce chondrogenic differentiation of human mesenchymal stem cell (hMSC) in agarose[non-biogradeable material] culture. Of the possible biocompatible materials available for tissue engineering, fibrin is a natural regenerative scaffold, which possesses several desired characteristics including a controllable degradation rate and low immunogenicity. The objective of the present study was to determine the capability of fibrin gels for supporting chondrogenesis of hMSCs under cyclic compression. To optimize the system, three concentrations of fibrin gel (40, 60, and 80 mg/mL) and three different stimulus frequencies (0.1, 0.5, and 1.0 Hz) were used to examine the effects of cyclic compression on viability, proliferation and chondrogenic differentiation of hMSCs. Cyclic compression (10% strain) at frequencies >0.5 Hz and gel concentration of 40 mg/mL fibrinogen appears to maintain cellular viability within scaffolds. Similarly, variations in gel component concentration and stimulus frequency can be modified such that a significant chondrogenic response can be achieved by hMSC in fibrin constructs after 8 h of compression spread out over 2 days."

"Aggrecan expression was upregulated in all compressed samples compared to controls regardless of gel concentration or stimulus frequency applied"

"only the lowest concentration gels (1.0× gel concentration) present a significant upregulation of this gene’s expression when compared to its control."

"Only the 1 Hz[versus 0.5 and 0.1Hz] stimulated cells present an increased expression of the collagen II gene when compared to the control samples"

Height Increasing by Inhibiting Fra-1?

LSJL may have an effect on Fra-1.  No change in expression over 2 fold or under 0.5 fold was observed but a change was expected.

Fra-1 governs cell migration via modulation of CD44 expression in human mesotheliomas

CD44 encourages chondrogenesis so inhibition is bad.

"Silencing of Fra-1, a component of the dimeric transcription factor, activator protein-1 (AP-1), inhibits mRNA expression of c-met and cd44 in rat mesothelioma cells and is causally linked to maintenance of the transformed phenotype. In a panel of human MM cells, Fra-1 mRNA expression in MM is complex and regulated by extracellular signal-regulated kinase (ERK1, ERK2), Src, and phosphatidyl-inositol-3-kinase (PI3K) pathways in a tumor-specific fashion. Cell lines with PI3K-dependent Fra-1 expression were SV40 positive and expressed the lowest basal Fra-1 levels. Levels of Fra-1 expression correlated with amounts of CD44 expression that were greater in simian virus 40 negative (SV40-) MM cells. Using dominant negative (dn), short hairpin (sh) and small interference (si) RNA constructs, we next demonstrate that expression of CD44, the principal hyaluronic receptor in MMs, correlates with Fra-expression in both simian virus 40 positive (SV40+) and SV40- MMs. Both Fra-1 and CD44 expression are linked to cell migration in SV40- MM cells. Lastly, in contrast to normal lung tissue, Fra-1 was expressed in 33 of 34 human MMs, and that all CD44+ tumors were SV40-. Fra-1 is associated with cell migration in human MMs and that Fra-1 modulation of CD44 may govern migration of selected MMs."

"CD44 is a type I transmembrane glycoprotein (85–200 kDa) and functions as the major cellular adhesion molecule for hyaluronic acid (HA), a component of the extracellular matrix (ECM)."

The below study confirms the inhibitory role of Fra-1 on chondrogenesis.

Fra-1/AP-1 impairs inflammatory responses and chondrogenesis in fracture healing.

"Transgenic overexpression of Fra-1, a component of the transcription factor activator protein-1 (AP-1), in various tissues progressively and globally enhances bone formation. We created a transverse fracture of the mouse tibial diaphysis and examined fracture healing radiologically, histologically, and immunologically. Strikingly, fracture union was delayed even though the bone formation rate in callus was higher in Fra-1 transgenic (Tg) mice. In these mice, chondrogenesis around the fracture site was impaired, resulting in accumulation of fibrous tissue, which interferes with the formation of a bony bridge across the callus. Immediately after fracture, induction of the inflammatory mediators TNF-alpha, interleukin (IL)-6, and Cox-2{LSJL downregulates Cox-2 and TNF-alpha and upregulates IL-6} was significantly suppressed in Fra-1 Tg mice followed, by the reduced expression of Sox-9 and BMP-2. Because serum prostaglandin E(2) (PGE(2)) levels were dramatically low in these mice, we administered PGE(2) to the fracture site using a slow-release carrier. The accumulation of fibrous tissue in Fra-1 Tg mice was significantly reduced by PGE(2) administration, and chondrogenesis near the fracture site was partially restored. The Fra-1-containing transcription factor AP-1 inhibits fracture-induced endochondral ossification and bony bridge formation presumably through suppression of inflammation-induced chondrogenesis."

So LSJL  increased Fra-1 expression based on the effects on TNF-alpha and COX-2.

"Fos family proteins, such as c-Fos, Fra-1, Fra-2, and FosB, heterodimerize with Jun proteins to form the dimeric transcription factor activator protein-1 (AP-1)"<-LSJL upregulates c-Fos.

Green tea increases Fra-1 levels according to Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes.

Estrogen induces Fra-1 according to this study, Estrogen-induced expression of Fos-related antigen 1 (FRA-1) regulates uterine stromal differentiation and remodeling.

According to this study C-Fos can also inhibit chondrogenic differentiation

Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene.

"ATDC5 chondrogenic cells, undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. We constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation.  Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation."

This is only ATDC5 cells, c-fos may have a different effect in HBMMSC's.

So decreasing c-Fos levels will also decrease Fra-1 levels and both these decreases will enhance chondrogenesis.

MS-275, a benzamide histone deacetylase inhibitor, prevents osteoclastogenesis by down-regulating c-Fos expression and suppresses bone loss in mice. states that MS-275 may be a potential c-Fos inhibitor.
BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis.

"Both BMP-7 and BMP-2 induced C3H10T1/2 cells [murine mesenchymal stem cell line] to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9{up in LSJL}, Msx 2, and c-fos{up in LSJL} during the periods of chondrogenic differentiation after which point their expression decreased. Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. The murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. The induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism."

"Depending on the concentration used, 5-azacytidine stimulated C3H10T1/2 to differentiate into myotubes, adipocytes, or chondroblasts"  5-azacytidine interferes with DNA methylation which can silence gene expression.

"In the high-dose BMP-7 treated cells, type II collagen expression increased dramatically and reached peak expression by 4 days after the initiation of BMP-7 treatment and was maintained at this high level until day 8, after which it sharply declined. The low-dose BMP-7 treated cells maintained their type II collagen expression longer than the untreated cells but never obtained the same maximal level of expression seen in the 250 ng/ml dose group. "

"C-fos exhibited almost no expression in the control cultures but showed a very strong peak of expression in the BMP-7 treated groups during the period of maximal chondrogenic expression. Fra 2 expression was initially high in all groups of cultured cells with a slight increase in expression over time in all groups. Fra 1 was also expressed initially by all groups but decreased to near absent levels by day 2"

Shear- and compression-induced chondrocyte transcription requires MAPK activation in cartilage explants.

"Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. We applied dynamic shear to bovine cartilage explants. We measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1-24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage."

"In cartilage explants, static compression can induce the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and p38, and dynamic compression can induce ERK activation. In isolated chondrocytes, fluid shear activates ERK1/2"

"The increase in the phosphorylated ERK1/2 level reached a maximum value at 3% shear strain and slightly decreased at 4.5% shear strain; therefore time course experiments were performed at the 3% shear strain, 0.1 Hz condition. Interestingly, there was a dramatic increase in the phosphorylated ERK1/2 level even at 0% compression over the free swelling condition"

"The mechano-induced up-regulation of transcription factors Sox9, c-Fos, and c-Jun was partially suppressed by U0126[ERK inhibitor] under all loading conditions"

"p38 signaling contributes toward transforming growth factor-β-stimulated proteoglycan synthesis and chondrocyte proliferation (36, 37), and ERK1/2 is activated by insulin-like growth factor-1, which strongly promotes anabolic chondrocyte behavior"

Fos/AP-1 proteins in bone and the immune system.

"Induction of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) signals by activated T cells and subsequent activation of the key transcription factors Fos/activator protein-1 (AP-1), NF-kappaB, and NF for activation of T cells c1 (NFATc1) are in the center of the signaling networks leading to osteoclast-mediated bone loss. Conversely, nature has employed the interferon system to antagonize excessive osteoclast differentiation, although this counteracting activity appears to be overruled under pathological conditions."

"Mesenchyme-derived chondrocytes and osteoblasts are able to rebuild the resorbed bone by producing the extracellular matrix that eventually gives rise to the known mineralized skeletal elements and also to joints and tendons"

"the Fos proteins (Fos, FosB, Fra-1, and Fra-2) can only heterodimerize with members of the Jun family, the Jun proteins (Jun, JunB, and JunD) can both homodimerize and heterodimerize with Fos members to form transcriptionally active complexes"

"Chimeric mice obtained from Fos-overexpressing embryonic stem cells develop chondrogenic tumors, implying a function of Fos in chondrogenesis. Surprisingly, Fos overexpression in an in vitro model of chondrogenesis inhibited the differentiation of chondrocytes. Ectopic Fos expression from a ubiquitous promoter in transgenic mice has no noticeable effects on cell differentiation in most organs, but it results in the specific transformation of osteoblasts leading to osteosarcoma formation"

"NFATc1 was originally described as a Fos target gene in osteoclastogenesis, but recent evidence has shown that it is also regulated by NF-κB."

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones.

"The highest levels of TGF-beta mRNA [are] associated with the growth plates. This mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. the highest levels of c-fos mRNA were detected in epiphyseal cartilage. [The] two cell types with high levels of c-fos expression [were] the chondrocytes bordering the joint space and the osteoclasts of developing bone."

"Deregulated expression of c-fos under the metallothionein promoter in transgenic mice was found to specifically interfere with the development of long bones"

"TGF-B [may] not [be] associated with the chondrocytes of the growth plate"<-this study is from 1988 though.

"osteoclasts also contain high levels of c-fos transcripts"
Distribution and expression of mRNAs for the proto-oncogenes c-fos and c-jun in bone cells in vivo.

"Femurs of 4-week-old rats were examined. C-fos and c-jun exhibited similar distribution in growth plate and bone tissue. Expression of c-fos and c-jun mRNAs in growth plate was observed in the proliferative zone and partly in the upper layer of the hypertrophic zone. In spongy bone, high expression of c-fos and c-jun mRNAs was observed in the osteoblast cytoplasm. There was little expression in bone lining cells. In the bony trabeculae, slight expression of c-fos and c-jun was observed in the premature osteocytes situated close to the bone surface, but no expression was detected in osteocytes that possessed relatively large lacunae in the center of the trabeculae. C-fos and c-jun were also slightly expressed in osteoclasts. c-fos and c-jun are involved in regulating chondrocyte proliferation as immediate early genes, and may also be involved in the gene expression of bone matrix proteins as transcription factor (AP-1) in vivo. Strong expression [of c-Fos and c-Jun]  in osteoblasts but hardly any expression at all in bone lining cells [suggests] that these genes are involved in oteoblast activation."

We need to find a selective c-Fos inhibitor that doesn't inhibit the NF-kappaB pathway also.  However there have been studies that report a stimulatory effect of c-Fos on chondrogenesis.