Tuesday, February 14, 2012

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

"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"

"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."

"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.

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