Previously, we discussed some methods that may govern cellular senescence and in turn growth plate senescence including DNA Methylation and telomere length. Endochondral ossification stops as a result of cellular senescence in the growth plate. DNA Methylation and telomere length may regulate senescence in the stem cells but alkaline phosphatase may determine the terminal chondrogenic differentiation in growth plate chondrocytes. Studying growth plate alkaline phosphatase activity may be a key in curing dwarfism and in increasing height in normal individuals.
F-spondin regulates chondrocyte terminal differentiation and endochondral bone formation.
"F-spondin [is] an extracellular matrix protein of osteoarthritic cartilage [expressed] during chondrocyte maturation in embryonic growth plate cartilage. In chick tibia, F-spondin expression localized to the hypertrophic and calcified zones of the growth plate. F-spondin inhibited (∼35%), and antibodies to F-spondin increased (∼30%) longitudinal limb growth relative to untreated controls[inhibiting F-spondin will increase your height]. Induction of chondrocyte maturation, by retinoic acid (RA) or transforming growth factor (TGF)-β treatment led to a significant upregulation of F-spondin. F-spondin transfection increased mineral deposition, alkaline phosphatase (AP) and matrix metalloproteinase (MMP)-13 mRNA levels), and AP activity following RA stimulation, compared to mock transfected controls. Using AP as a differentiation marker we then investigated the mechanism of F-spondin promaturation effects. Blocking endogenous F-spondin via its thrombospondin[we've linked thrombospondin to height increase before] (TSR) domain inhibited RA induced AP activity 40% compared to controls. The stimulatory effect of F-spondin on AP expression was also inhibited following depletion of TGF-β from culture supernatants. Our findings indicate that F-spondin is expressed in embryonic cartilage, where it has the capacity to enhance chondrocyte terminal differentiation and mineralization via interactions in its TSR domain and TGF-β dependent pathways."
F-spondin increases Alkaline Phosphatase activity in growth plate chondrocytes and encourages them to differentiate into bone cells. Inhibiting F-spondin can make you taller.
"F-spondin treatment was found to increase both type II collagen degradation and MMP-13 secretion"
"inhibition of F-spondin by antibody treatment caused a reduction in the number of hypertrophic chondrocytes"
"in the presence of RA, F-spondin enhances terminal differentiation and mineralization, but not induction of hypertrophy, of chick sternal chondrocytes"<-So F-spondin only has negative effects with Retonoic Acid?
"F-spondin [inhibitor] treated limbs revealed increased numbers of hypertrophic chondrocytes in the growth plate cartilage adjacent to the mineralized core"<-F-spondin reduces the number of hypertrophic chondrocytes.
"F-spondin mRNA levels, along with MMP13, were highest within the calcified region. "
"addition of F-spondin to cartilage explant cultures was found to increase active TGF-β levels in culture supernatants and regulate synthetic activity."<-This is something we'd expect to increase height not decrease height. However, "Late hypertrophic chondrocytes produce more TGF-β than early hypertrophic chondrocytes and the percentage of TGF-β that is activated increases as the chondrocyte nears terminal differentiation." So the greater amount of TGF-Beta can be due to more terminal differentiating chondrocytes than F-spondin upregulating TGF-Beta.
"treatment of embryonic chick chondrocytes with TGF-β2 for 24 h in a serum-free alginate culture system led to dose dependent increases in expression of hypertrophic markers AP (~1.3- to 4.3-fold) and MMP-13 (~3.5- to 30-fold), as well as F-spondin (~2.4 to 5-fold)"
"Addition of exogenous recombinant TGF-β increases the expression of MMP-13 and AP as well as F-spondin. Thus, late-stage hypertrophic chondrocytes may respond to activated TGF-β by further progression toward terminal differentiation. F-spondin is part of a positive feedback loop that accelerates this process, ensuring terminal differentiation of hypertrophic chondrocytes and subsequent endochondral ossification via induction of MMP-13 and further activation of TGF-β"<-so inhibiting F-spondin will knock out the positive feedback loop that causes terminal differentiation.
Since only alkaline phosphatase and MMP13 were monitored it's possibly that other genes affected by F-spondin are the ones mediating the height decreasing effect.
The role of pyrophosphate/phosphate homeostasis in terminal differentiation and apoptosis of growth plate chondrocytes.
"Extracellular inorganic phosphate (P(i)) concentrations are the highest in the growth plate just before the onset of mineralization. P(i) not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular P(i) stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular P(i) on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na(+)/P(i) cotransporters, and ultimately P(i) uptake. A high extracellular P(i) concentration was required for the stimulation of apoptosis, whereas lower P(i) concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular P(i) on terminal differentiation events. On the other hand, increasing the local extracellular P(i) concentration by overexpressing ANK, a protein transporting intracellular PP(i) to the extracellular milieu where it is hydrolyzed to P(i) in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PP(i) to P(i), ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PP(i) inhibiting and extracellular P(i) stimulating hypertrophic and terminal differentiation events, a precise regulation of PP(i)/P(i) homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes."
"APase, hydrolyzes extracellular PPi into Pi. Extracellular PPi concentrations in the growth plate are mainly regulated by two proteins, the phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1) and the progressive ankylosis protein (ANK). PC-1 is an enzyme that hydrolyzes extracellular adenosine triphosphate, thereby producing PPi. ANK is a transmembrane protein that transports intracellular PPi to the extracellular milieu. Extracellular Pi–induced effects on skeletal tissue cells and other cells are dependent on Pi entry into cells."
"The primary mechanism for extracellular Pi entry through the cell membrane is via a family of Na+-dependent Pi transporters."<-NPT3 for growth plate chondrocytes. So this is another means to how sodium transporters can influence height.
"increasing concentrations of extracellular Pi resulted in further decreases in the mRNA levels of type II and X collagen with 8mM Pi being the most effective in decreasing mRNA levels of type II and X collagen"<-evidence shows that type II collagen is good for height so increasing concentrations of extracellular Pi may be bad for height.
"PFA treatment[a sodium-potassium co-transporter] resulted in decreased mRNA levels of APase, MMP-13, osteocalcin, and runx2 compared to the mRNA levels of these genes in growth plate chondrocytes treated with various concentrations of extracellular Pi in the absence of PFA, whereas mRNA levels of type II and X collagen increased"
"Extracellular Pi concentrations in the growth plate increase when growth plate chondrocytes undergo hypertrophic differentiation and reach the highest levels just before mineralization of the extracellular matrix starts"<-So lowering these levels by exogenous means may keep growth plate chondrocytes from terminal differentiation longer and thus keep you growing taller longer.
So reducing extracellular inorganic phosphate may be a means of growing taller in active growth plates.
24R,25-Dihydroxyvitamin D3, lysophosphatidic acid, and p53: a signaling axis in the inhibition of phosphate-induced chondrocyte apoptosis., states
Like with Estrogen, an optimal amount of phosphate levels in the growth plate is required. Levamisole is an Alkaline Phosphatase inhibitor and may be worth investigating. Teriparatide may be the best solution...
Parathyroid hormone 1-34[Teriparatide] inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats.
"Parathyroid hormone 1-34 (PTH[1-34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone-related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1-34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA).
METHODS: We studied the effect of PTH(1-34) on human articular chondrocytes with azacytidine (azaC)-induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees.
RESULTS: AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3-10 days of treatment with 10 nM PTH(1-34). SOX9 expression was not changed by either azaC or PTH(1-34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1-34) treatment did not reverse this effect. Furthermore, PTH(1-34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats.
CONCLUSION: Our findings indicate that PTH(1-34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA."
"In this study, we also found that PTH(1–34) treatment of human articular chondrocytes without azaC induction of terminal differentiation and of rat knee joints without OA did not significantly alter the expression of type II collagen, type X collagen, AP, aggrecan, or GAG in articular chondrocytes. This finding suggests that PTH(1–34) may not affect chondrocyte functions in healthy joints. No significant difference in the tested markers was found in the contralateral control knees between the OA and OA plus PTH groups, indicating that PTH(1–34) may have an effect on articular cartilage locally, and only on OA-affected cartilage."
So, perhaps Teriparatide may not increase growth in normal growth plates.
MSX2 stimulates chondrocyte maturation by controlling Ihh expression.
"A homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. We examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression."
Too much BMP-2 is bad and results in premature ossification of growth plate chondrocytes. But BMP-2 is necessary for chondrocyte hypertrophy and for normal growth.
Collagen/annexin V interactions regulate chondrocyte mineralization.
"Physiological mineralization in growth plate cartilage is restricted to terminally differentiated chondrocytes. Types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization."
Now, just because mineralization is enhanced doesn't mean growth is reduced. The only study that showed an increase in growth was inhibiting F-spondin. Therefore, the best tool for height increase and curing conditions such as dwarfism is to research these F-spondin inhibitors and antibodies.
Here's a study linking Alkaline Phosphatase to c-Fos. LSJL upregulates c-Fos as well.
The function of heterodimeric AP-1 comprised of c-Jun and c-Fos in activin mediated Spemann organizer gene expression.
"Overexpression of heterodimeric AP-1 comprised of c-jun and c-fos (AP-1(c-Jun/c-Fos)) induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin[BMP-2 is good for height growth, however LSJL does not upregulate c-Jun]. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene[BMP-4 is an inducer of chondrogenesis]. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes.
AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression in downstream of activin signal during early [an amphibian organism] embryogenesis."
" functional cooperatives of between Smad3 and AP-1 in TGFβ signaling and a crucial role of Smad3 in the regulation of chordin"
"Smad3 significantly induced AP-1 activity"