Tuesday, June 19, 2012

Grow taller by manipulating HMGA2?

HMGA2 is a key gene modulating human height with it's expression and it's inhibitor let-7(CDK6 gene) observed in several studies as modulating stature.

HMGA2: A pituitary tumour subtype-specific oncogene?

"The Hmga2 gene has a critical role in the control of body growth and adipocytic cell proliferation and differentiation. Hmga2-null mice showed a pygmy phenotype with a decreased body size of 20% in heterozygous and 60% in homozygous mice, and a drastic reduction of the fat tissue, while transgenic mice expressing a truncated Hmga2 without the 3′ UTR demonstrate gigantism. alleles of SNPs linked to HMGA2 have been found associated with human height, and a germline chromosomal inversion that produces a truncated HMGA2{up in LSJL} gene was identified in a boy with severe overgrowth.
HMGA2-linked SNPs most strongly associated with height lie in the HMGA2 3′ UTR, which is bound by the microRNA (miR) Let-7. The height-influencing genetic event linked to these SNPs may possibly influence Let-7 binding and its consequent downregulation of HMGA2 protein expression."

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Ignore the pituitary transformation part of this but you can see how HMGA2 influences the cell cycle and this may be how HMGA2 alters height growth.

In vivo modulation of HMGA2 expression.

"A phenotypic analysis performed on homozygous mutants at the pygmy locus showed that the adults are 40% of the size of wild-type litter-mates whereas adult heterozygotes are 80% of the size of wild-type litter-mates."

"Hmga2 negatively regulates p16Ink4a and p19Arf"

"HMGA2 expression is not detected in normal adult tissues, low level of HMGA1 expression has been observed in adult tissues of both humans and mice"

Inhibiting let7 may inhibit its binding to HMGA2 allowing it to be expressed in adult tissues.

"targeted knock-out mouse that lacked exons 2 and 3 of Hmga2 [is] referred to as the Hmga2 null mouse (Hmga2−/−). Hmga2−/− mice had the same phenotype that was observed in the pygmy mice and the growth retardation in these mice resulted from a decrease in mesenchymal cell proliferation"

Lin28 is an inhibitor of let-7 so increasing levels of that may allow for HMGA2 expression.

Metformin may antagonize Lin28 and/or Lin28B activity, thereby boosting let-7 levels and antagonizing cancer progression.

"The let-7 family of microRNAs has cancer suppressor activity. Markedly reduced levels of let-7 are largely responsible for cancer stemness.  Metformin opposes cancer cell stemness [and boosts] let-7a levels in cancer stem cells. Metformin may reflect AMPK-mediated{AMPK Beta-2 subchain is downregulated by LSJL} inhibition of the expression or activity of Lin28/Lin28A, proteins which act post-transcriptionally to decrease the levels of all let-7 family members[don't take metformin if you want to grow taller]. The transcription of Lin28B{upregulated by LSJL} is promoted by NF-kappaB and by Myc[stimulating both NF-kappaB and Myc are options]].  [Antagonists for] NF-kappaB or Myc activity [include] salsalate, antioxidants, tyrosine kinase and cox-2 inhibitors[thus you may want to increase tyrosine kinase and COX-2 to grow taller], ribavirin, vitamin D, gamma-secretase inhibitors (when available), and parenteral curcumin may have some utility in this regard."

"The PI3K-Akt-GSK3β pathway downstream from most tyrosine kinase receptors can stimulate Myc transcription by promoting nuclear localization of beta-catenin, and also boost EMT by blocking the GSK3β-mediated nuclear export and proteasomal degradation of Snail. And mTOR activation by growth factors (opposable by metformin) can boost the translation of Myc mRNA"<-methods to increase Myc

"Myc mRNA is one of those so-called “weak” mRNAs whose translation can be boosted by interaction with eIF4E. mTOR activity amplifies the availability of free eIF4E by phosphorylating and thereby disabling 4EBP-1, which functions to bind and sequester eIF4E."<-so other activities that increase free eIF4E may boost height as well.

"STAT3 [induces] Myc"

"Notch1 directly promotes transcription of Myc"

"let-7 [sustains] its own expression by antagonizing Lin28, Ras, and Myc translation"<-So we have to find creative ways to inhibit let-7

"Let-7 expression can be globally but specifically reduced via increased activity of Lin28/Lin28B"

let-7/HMGA2/Lin28 are way more important to research than CNP.  Several genes in height growth gene association studies are related to these.  Find homeopathic remedies or supplements that decrease let-7 or increase HMGA2/Lin28.

This page claims that it's Astragalus increases Lin28 levels and that it's Astragalus has been specifically engineered to increase Lin28(I couldn't find any studies on Astragalus and Lin28).  The site does not seem legit though.

This scientist explains that basketball players may be lin28 phenotypes.

TTP is a target to increase Lin28/decrease let-7/increase HMGA2.

Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28.

"Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. let-7 microRNA has emerged as a significant factor in [cell growth] suppression{anything that causes cell growth makes cancer grow faster but it doesn't cause cancer}. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. TTP promotes an increase in expression of mature let-7{so we can decrease TTP to decrease Let-7}, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. [TTP-mediates] inhibition of Lin28. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis."

And this study says MAPK represses TTP:

"mRNA turnover is a critical step in the control of gene expression. In mammalian cells, a subset of mRNAs regulated at the level of mRNA turnover contain destabilizing AU-rich elements (AREs) in their 3' untranslated regions. These transcripts are bound by a suite of ARE-binding proteins (AUBPs) that receive information from cell signaling events to modulate rates of ARE mRNA decay. A key destabilizing AUBP, tristetraprolin (TTP), is repressed by the p38 mitogen-activated protein kinase (MAPK)-activated kinase MK2 due to the inability of phospho-TTP to recruit deadenylases to target mRNAs. TTP is tightly associated with cytoplasmic deadenylases and promotes rapid deadenylation of target mRNAs both in vitro and in cells. TTP can direct the deadenylation of substrate mRNAs when tethered to a heterologous mRNA, yet its ability to do so is inhibited upon phosphorylation by MK2. Phospho-TTP is not impaired in mRNA binding but does fail to recruit the major cytoplasmic deadenylases. phosphorylation of TTP by MK2 primarily affects mRNA decay downstream of RNA binding by preventing recruitment of the deadenylation machinery. TTP may remain poised to rapidly reactivate deadenylation of bound transcripts to downregulate gene expression once the p38 MAPK pathway is deactivated."

"Phosphorylation of TTP by the p38-activated kinase MK2 promotes 14-3-3 association and inhibits the ability of TTP to trigger the deadenylation of tethered mRNA in cells by preventing the recruitment of cytoplasmic deadenylases. In contrast, phosphorylation does not affect the ability of TTP to bind mRNA. "<-so it's possible that MAPK doesn't impact the ability of TTP to stimulate let-7.

I shot an email to a writer of the study but Lin28 is a protein not mRNA.

According to RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation., L1TD1 could increase the effect of Lin28.

"A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-kappaB, Lin28{Lin28B is up in LSJL}, let-7, and IL-6{up in LSJL}. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD{down in LSJL} tumor suppressors, leading to increased NF-kappaB activity required to maintain the transformed state. STAT3-mediated regulatory circuits are required for [transformed states] in diverse cell lines. STAT3 is not only a downstream target of IL-6 but, with miR-21, miR-181b-1, PTEN, and CYLD, is part of the positive feedback loop that underlies the epigenetic switch that links inflammation to cancer."

So we have to study this loop to see if inflammation can induce Lin28.

"Lin28 is a direct target of NF-κB"->So NF-kappaB may actually increase height growth as evidenced by the studies on proepithelin.

"The transcription factor NF-κB [may be] required for inhibition of the Let-7 microRNA family but this regulation is indirect and involves Lin28 as an intermediary protein"<-so you may be able to get around NF-kappaB by directly stimulating Lin28 in other ways.

"Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop."

"Phosphorylation of STAT3, a downstream target of IL6, is inhibited by the addition of let-7a or by inhibition of Lin28B"<-phosphorylation of STAT3 may be a way to increase height.

"IL6 treatment inhibits let-7a microRNA expression in a manner that depends upon NF-κB"

More on TTP:

Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2).

"TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. TTP contributes to the NF-κB/JNK balance. MAP 3-kinase MEKK1 [is a] TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination[So MEKK1 is another target for height increase]. TTP no longer affects NF-κB activity but promotes the activation of JNK.  Upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades."

So we don't want MEKK1 as we want TTP to go down the NF-kappaB cascade.  So inhibiting MEKK1 could help increase height.

"The MAPK p38α induces TTP phosphorylation, traceable by a “molecular weight shift” of the protein in Western blots, inactivating TTP mRNA binding and degrading ability, but in contrast, p38α did not block TTP function toward NF-κB"<-Thus why we want MAPK38 to be activated and not MEKK1.

"coexpression of MEKK1, which has not been described as TTP kinase yet, could even counteract TTP inhibition of p65-induced NF-κB promoter activity"

"TGFβ acts on the control of NF-κB: TNFα stimulation of TGFβ pretreated cells results in restricted NF-κB target gene expression caused by impaired p65 nuclear translocation"<-Thus we might have to upregulate NF-kappaB to get around the negative feedback that TGF-Beta exerts on NF-kappaB.

Inhibiting PKCDelta is a height increase target as well according to Inhibition of protein kinase Cdelta reduces tristetraprolin expression by destabilizing its mRNA in activated macrophages.

It's possible that you may want inflammation when performing LSJL to induce NF-kappaB decrease in let-7. This would involve longer durations of LSJL to induce more inflammation.

Overexpression of HMGA2-LPP fusion transcripts promotes expression of the alpha 2 type XI collagen gene.

"In a subset of human lipomas, a specific t(3;12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the alpha 2 type XI collagen gene (Col11a2).  HMGA2-LPP and COL11A2 were co-expressed. either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the Col11a2 promoter in HeLa cells, while the C-terminal LPP did not. HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains."

"only lipomas associated with HMGA2-LPP contained detectable COL11A2 transcripts"

"overexpression of the N-terminal DNA binding domains of HMGA2 in transgenic mice or constitutionally rearranged human results in overgrowth and lipomatosis"

"full-length HMGA2 can activate Col11a2 promoter when overexpressed. HMGA2 modulates the activity of the DNA repair gene ERCC1{down in LSJL} and of the cyclin A gene by binding to these genes"

"recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro"

"statistically significant increase in proliferation could be detected in chondrocytes treated with either HMGA2-A peptides at both 10 μM (1.94x increase) and 50 μM (1.88x increase), or wild-type HMGA2 protein (10 μM, 2.1x increase){this was the only dose of HMGA2 used}." 

"LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras{down in LSJL}, Igf2bp1 and Hmga2{up} are known regulators of mammalian body size and metabolism. we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. These transgenic mice [had] increased glucose metabolism and insulin sensitivity."

"Lin28a Tg mice possess increased body size, a phenotype associated with genetic variation in the human LIN28B locus"

"Lin28a has been shown to enhance protein translation of Igf2, whose loss of imprinting causes a human overgrowth disorder called Beckwith-Wiedemann Syndrome (BWS). We found a ~20-fold increase of Igf2 mRNA in liver and a 2-fold increase in muscle. To determine if Igf2 was driving overgrowth, we crossed Lin28a Tg females to Igf2 null males and noted that Lin28a Tg mice lacking Igf2 were still overgrown "

"LIN28B alleles associated with later menarche were linked to taller stature"

Genetic variation in candidate genes like the HMGA2 gene in the extremely tall.

"The HMGA2 gene SNP [is] significantly associated with tall stature. Carrying the HMGA2 (rs1042725) C allele significantly increased the odds of being tall . In addition, controls with one or two copies of the C allele were significantly taller than controls carrying the TT genotype.
A common polymorphism in the HMGA2 gene is not only associated with height variation in the general population but also plays an important role in one of the extremes of the height distribution."

"The VDR gene encodes a nuclear receptor for 1,25-dihydroxyvitamin D. One study observed significant linkage for a functional SNP in its gene that may be responsible for 34% of idiopathic short stature cases"

"In humans, each copy of the C allele of this SNP was associated with an increase of about 0.4 cm in height in the general population"

Insights into the regulation of a common variant of HMGA2 associated with human height during embryonic development.

"genome-wide SNP studies revealed a significant association of rs1042725 genotypes CT and CC in the 3' UTR of HMGA2 with human height. HMGA2 expression levels during prenatal development might be a critical factor that contributes to the height phenotype. The rs1042725 genotype is unlikely to affect HMGA2 levels in pluripotent human embryonic stem cells (hESCs). hESCs in the inner cell mass of blastocysts are most likely not involved in determining the human height phenotype associated with this SNP. miR-196b as a candidate microRNA that could contribute to SNP-specific expression of HMGA2 during human prenatal development."

"HMGA2 is highly expressed in pluripotent human ES (hES) cells and contributes there to cell proliferation control. Variations in HMGA2 expression levels during these earliest stages of human development might influence adult stature. Based on the location of rs1042725 in the 3’UTR of HMGA2, microRNAs (miRs) play a role in HMGA2 regulation"

C-Terminal allele is the allele that's good for height.  You want CC.

"miR-196b [is] a candidate that specifically targets the C-allele of rs1042725 in a MRE and compromises expression of a reporter gene. miR-196b affects expression of Hoxb8 and Hoxa7 genes, which are involved in vertebrate limb formation during embryonic development"

"the presence of C-alleles at rs1042725 will lead to’reduced HMGA2 levels when compared with cells carrying T-alleles. Significantly increased body height is usually observed in mice and man when the HMGA2 gene is constitutively overexpressed during embryonic development and, later, in adult tissues of the body due to germline genomic alterations{so if LSJL can upregulate HMGA2 in the bone it could still help for adult height growth}. It is important to recognize that this is clearly an abnormal situation, which does not reflect normal development in a wild-type HMGA2 background when the temporal control of HMGA2 expression levels in, for example, specific progenitor stem cells might impact on body height.   Reduction of HMGA2 levels in combination with changes in expression patterns of other genes, such as certain Hox genes, might eventually lead to an increased body height. HMGA2 levels decrease during ESC differentiation, while miR-196 expression increases"

LIN28A Is a Suppressor of ER-Associated Translation in Embryonic Stem Cells.

"LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins."

"Lin28a is highly expressed in embryonic stem cells (ESCs) and [is] one of the four factors that convert fibroblasts into induced pluripotent stem cells.  In [mice], Lin28a deficiency caused undergrowth in early stages of development, whereas its ectopic expression induced overgrowth and delayed the timing of puberty"

"LIN28 binds to the primary transcript of let-7 (pri-let-7) and prevents its processing by RNase III DROSHA. In the cytoplasm, it interacts with the precursor form of let-7 (pre-let-7) and interferes with pre-let-7 processing. LIN28 recruits TUTase 4 (ZCCHC11) to induce oligo-uridylation of pre-let-7, which effectively blocks DICER processing and facilitates degradation of the RNA. Although LIN28B is localized mainly in the nucleolus and interferes with nuclear processing, LIN28A is found mostly in the cytoplasmic compartment and acts in concert with TUTase 4. LIN28 homologs commonly have two types of RNA binding domains: a cold shock domain and a cluster of two CCHC-type zinc finger motifs. The “GGAG” sequences in the terminal loop of let-7 precursors serve as the binding site for the zinc finger domains that are critical for let-7 regulation"

"LIN28A can bind to and enhance translation of certain mRNAs such as Igf2 in differentiating myoblasts and Oct4 in ESCs "

"In mESCs, the let-7 family is likely to be the only functional miRNA target of LIN28A."

"LIN28A is a positive regulator of translation for mRNAs Igf2, cyclin A, cyclin B, histone 2a, and Oct4"

"The mRNAs coding LAMP1, EpCAM, and E-cadherin interact with LIN28A"

" LIN28A targets mRNAs for translational repression."  LIN28A represses all of the let7 family as verified by LIN28A knockdown studies.

12q14 microdeletion associated with HMGA2 gene disruption and growth restriction.

"The 12q14 microdeletion syndrome is a rare condition that [is] characterized by pre- and postnatal growth restriction, proportionate short stature, failure to thrive, developmental delay, and osteopoikilosis. Microdeletions within this region have ranged in size from 1.83 to 10.12 Mb with a proposed 2.61 Mb smallest region of overlap containing the LEMD3, HMGA2, and GRIP1 genes. [We identified] a 12q14 microdeletion in a female child presenting with proportionate short stature, failure to thrive, and speech delay. The genomic loss (minimum size 4.17 Mb, maximum size 4.21 Mb) contained 25 RefSeq genes including IRAK3, GRIP1, and the 3' portion of the HMGA2 gene.  partial deletion of HMGA2 [is] associated with the 12q14 microdeletion syndrome.  LEMD3 deletions [associates] with the 12q14 microdeletion syndrome. [HMGA2 has a role in human growth]."

Short stature is proportional leading weight to the theory that HMGA2 increases height by transcriptional repression of embryonic development leading to longer embryonic development and thus greater height growth.  All cases of this genomic deletion had proportional short stature which leads credence to the theory that HMGA2 affects height at the embryonic stage.

Probing into the Biological Processes Influenced by ESC Factor and Oncoprotein HMGA2 Using iPSCs.

"The high mobility group AT-hook 2 (HMGA2) protein is a nonhistone chromatin factor normally expressed in ESCs and during early developmental stages.  We used iPSC formation in conjunction with exogenous human HMGA2 expression. Anatomical development and cell adhesion/differentiation processes are strongly affected by HMGA2.  Expression of key diabetes susceptibility genes is influenced by HMGA2, which revealed an interesting link to the recently indentified Lin28/let-7 pathway regulating mammalian glucose metabolism. HMGA2 is not involved in the regulation of telomerase gene expression. Tight regulation of intracellular HMGA2 levels is important both to maintain a pluripotent ESC state and to induce differentiation into certain cell lineages during later developmental stages."

"In many tumor cells, including tumor initiating cells, HMGA2 is re-expressed as a result of Lin28-mediated let-7 miRNA degradation"

"HMGA2 exerts effects during pre- and postnatal development. miR-196b [is] involved in SNP-specific regulation of HMGA2 expression, during prenatal development, which is linked to an increased body stature in humans"

"Expression of HMGA2 in fibroblasts, which do not express HMGA2, promotes cell cycle arrest"

HMGA2 transgene iPSCs expressed less Utf1, Nr5a2, Oct4, Nanog, and Sox2 versus controls.

"HMGA2 expression [does not] influence iPSC morphology or affect some of the pluripotency phenotypes, although a slight decrease in iPSC colony number was detected."

"HMGA2 knockdown in human ESC showed a down-regulation of Nes"

"Igf2bp2, Irs1 and PiK3ip1 were up-regulated in cells with high HMGA2 levels"

"Prdm9, Dcdc2, Fcgbp, Fam107b and Clgn were up-regulated in H1 and H2 [HMGA2 transgene] cell lines"

"HMGA2 expression levels [are] critical both for maintenance of pluripotency and differentiation."

It should be noted that EFGP transgene was silenced after passage 8.

Genes upregulated by H1 and H2(these cell lines had the most significant increase in HMGA2 levels) and were differentially regulated by all three cell lines H1-H3 also upregulated by LSJL:
Col6a2{up and down}


1 comment:

  1. I have to say these are quite amazing finds, wow.