Factors that affect LSJL Gene Expression

Previously, we found that the benefits of LSJL did not relate to the dynamic loading of chondrocytes.  Under dynamic loading of chondrocytes, the anabolic benefits seemed to be due to the upregulation of aggrecan, Type II collagen, and osteopontin whereas the anabolic benefits of Lateral Joint Loading seem to be due to an upregulation of genes involved in the PI3K pathway and TGF-Beta.  It would make sense for the anabolic response(the height increase) of LSJL be due to loading of the periosteal component(or fibrous capsule) of the bone as that is the part of the bone that is loaded differently in lateral versus vertical loading.

Two potential factors that modulate LSJL gene expression are loading site(more on the cartilage versus the epiphysis) and circulating levels of estrogen(how much estrogen is optimal).

Mechanical loading stimulates rapid changes in periosteal gene expression. 

"External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein beta-actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor-beta (TGF-beta). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc[Less Alkaline Phosphatase discourages fusion], which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF-beta and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading[This increase in TGF-Beta and IGF-1 can have positive benefits on cellular proliferation]. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation.  Increased periosteal cell proliferation [is] seen both in vivo and in vitro following mechanical loading."

Okay, since dynamic loading of chondrocytes didn't upregulate genes encouraging chondrogenesis like Sox9 but Periosteal loading increased TGF-Beta which encourages chondrogenesis a key part of Lateral Synovial Joint Loading is periosteal shear.  The periosteal shear increases TGF-Beta which encourages a chondrogenic lineage for MSCs.  The benefits seen under LSJL more closely match those of periosteal shear(which is induced by IFF) than that of dynamic compressive loading of chondrocytes.  So it is more likely that it's the fluid flow that causes the periosteal shear strain induces the LSJL benefits(which can induce height gain if the fluid flow increases hydrostatic pressure in the epiphysis) than as a direct result of a compressive loading(which don't seem to match gene expression pathways for LSJL in chondrocyte loading for example).

Therefore, in deciding versus loading the epiphysis versus the cartilage, loading the epiphysis is superior as you still get periosteal shear and you are more likely to cause bone deformation which increases hydrostatic pressure.

Estrogen also regulates the load on bone and this could affect what genes are expressed in lateral synovial joint loading.

Loading-related regulation of gene expression in bone in the contexts of estrogen deficiency, lack of estrogen receptor alpha and disuse.

"Loading-related changes in gene expression in resident cells in the tibia of female mice in the contexts of normality (WT), estrogen deficiency (WT-OVX), absence of estrogen receptor alpha (ERalpha(-/-)) and disuse due to sciatic neurectomy (WT-SN) were established by microarray. Total RNA was extracted from loaded and contra-lateral non-loaded tibiae at selected time points after a single, short period of dynamic loading sufficient to engender an osteogenic response. There were marked changes in the expression of many genes according to context as well as in response to loading within those contexts. In WT mice at 3, 8, 12 and 24 h after loading the expression of 642, 341, 171 and 24 genes, respectively, were differentially regulated compared with contra-lateral bones which were not loaded. Only a few of the genes differentially regulated by loading in the tibiae of WT mice have recognized roles in bone metabolism or have been linked previously to osteogenesis (Opn, Sost, Esr1, Tgfb1, Lrp1, Ostn, Timp, Mmp, Ctgf, Postn and Irs1, BMP and DLX5)[TGF-Beta1, Timp, MMP, IRS-1, and BMPs are compounds that can affect chondrocytes, so the osteogenic response of bone to Estrogen matters even if ER-alpha doesn't have a direct effect of chondrocytes or MSCs]. The canonical pathways showing the greatest loading-related regulation were those involving pyruvate metabolism, mitochondrial dysfunction, calcium-induced apoptosis, glycolysis/gluconeogenesis, aryl hydrocarbon receptor and oxidative phosphorylation. In the tibiae from WT-OVX, ERalpha(-/-) and WT-SN mice, 440, 439 and 987 genes respectively were differentially regulated by context alone compared to WT. The early response to loading in tibiae of WT-OVX mice involved differential regulation compared to their contra-lateral non-loaded pair of fewer genes than in WT, more down-regulation than up-regulation and a later response. This was shared by WT-SN. In tibiae of ERalpha(-/-) mice, the number of genes differentially regulated by loading was markedly reduced at all time points. These data indicate that in resident bone cells, both basal and loading-related gene expression is substantially modified by context. Many of the genes differentially regulated by the earliest loading-related response were primarily involved in energy metabolism and were not specific to bone."

"estrogen receptor (ER), specifically ERα, has been shown to be involved in bone cells' early responses to strain"<-Possibly chondrocytes and stem cells as well

"genetic variation in ERα is associated with different responses to load-bearing exercise"

"In mice when ERα is absent the adaptive response to artificial loading in vivo is attenuated"<-attenuated means reduced.  So lowering Estrogen Receptor-Alpha levels may reduce the effectiveness of joint loading modalities like LSJL.  Note this is Estrogen Receptor-Alpha not Estrogen itself.

"Seven of the 25 genes significantly up-regulated in Zhang and Yokota's LSJL gene expression study were also up-regulated in WT[the normal rats] loaded tibiae in our study. These were mas-related gpr (Mrgpr), tissue inhibitor of metalloproteinase 1 (Timp1), thrombospondin (Thbs), procollagen type III alpha1 (Col3a1), mast cell protease (Mmcp), matrilin 2 (Matn2) and proteoglycan 4 (Prg4)."<-Hyaluronan Synthase and MMP-3 are key differences, thus they are likely key to height growth

"OVX resulted in reduced expression levels of some genes previously associated with bone cells (integrin-alpha 1 (Itga1, − 2.1-fold), Itga4 (− 2.0-fold), heme oxygenase-1 (Hmox1, − 1.8-fold) and transforming growth factor beta 1 (Tgfb1, − 1.9-fold))[Estrogen deficiency resulted in a reduction of TGF-Beta expression] and others (nobox oogenesis homeobox (Nobox, − 7.8-fold) and titin (Ttn, − 2.4-fold)). Expression of the genes for insulin receptor substrate-1 (Irs1), creb-binding protein (Crebbp) and bone morphogenetic protein-4 (Bmp4)[BMP-4 is a capable of inducing chondrogenic differentiation therefore it is good in terms of growing taller] was up-regulated in tibiae from WT-OVX mice (2.6-, 2.1- and 1.6-fold, respectively)."

So in Estrogen deficiency chondrogenic differentiation may be inhibited by the lack of TGF-Beta but on the other hand BMP-4 is present which can also induce chondrogenic differentiation.

For ER-alpha knockout mice:

"striatin (Strn, 10.1-fold), peroxisome proliferative activated receptor gamma coactivator 1 alpha (Ppargc1a, 2.1-fold), myocyte enhancing factor-2a isoform 1 (Mef2a, 1.8-fold), integrin-beta 6 (Itgb6, 2.1-fold), integrin-beta 4 (Itgb4, 1.6-fold), myogenic factor 6 (Myf6, 2.9-fold), connective tissue growth factor (Ctgf, 2.0-fold), Ttn (1.7-fold), osteocrin (Ostn, 1.7-fold), caveolin-1 (Cav1, 1.7-fold), Cav2 (1.7-fold) and Cav3 (1.5-fold)[Caveolin-3 inhibits myostatin so it's upregulation is good for height growth]. Among the genes which were down-regulated were low-density lipoprotein receptor-related protein 5 (Lrp5, − 1.8-fold)[LRP5 is a GSK3 Beta phosphorylater so it's downregulation is not anabolic], acid phosphatase 5, tartrate resistant (Acp5, − 1.7-fold) and Tgfb1 (− 1.8-fold)."

TGF-Beta is downregulated as is LRP5 so it would seem like knocking out ER-Alpha may be bad for height growth.

Sciatic neurectomy refers to removing the sciatic nerve.  The sciatic nerve removal group had the most interesting gene expression patterns.

"Wnt/β-catenin signaling (Lrp1, 1.8-fold), Lrp4 (1.8-fold), Wnt-inducible signaling protein 1 (Wisp1, 1.7-fold), Wisp2 (2.2-fold), secreted frizzled-related protein 1 (Sfrp1, 2.2-fold), Sfrp2 (1.8-fold), Sfrp4 (2.0-fold) and dickkopf-3 (Dkk3, 1.7-fold). Other genes of interest up-regulated in WT-SN mouse tibiae include insulin-like growth factor II receptor[increasing IGF-II receptor is actually a bad thing as it reduces circulating levels of IGF-II] (Igf2r, 2.4-fold), Ctgf (1.7-fold), growth differentiation factor 10 (Gdf10, 1.5-fold), fibroblast growth factor 8 (Fgf8, 1.5-fold), ERα (Esr1, 1.8-fold)[interesting that with no sciatic nerve Estrogen Receptor Alpha is increased], early growth response 1 (Egr1, 2.2-fold)[Egr-1 is also upregulated by LSJL], Myf6 (7.4-fold), periostin (Postn, 2.4-fold), ankyrin repeat domain 1 (Ankrd1, 7.6-fold), Ostn (1.7-fold), osteoglycin (Ogn, 2.5-fold), distal-less homeobox 5 (Dlx5, 1.8-fold), integrin-beta 4 (Itgb4, 1.8-fold), tenascin (Tnn, 2.1-fold) and Bcl-2/E1B 19-kDa interacting protein 3 (Bnip3, 2.1-fold). Five genes coding for matrix metalloproteinases (MMPs) were also up-regulated. A large number of genes involved in adhesion were markedly up-regulated in the tibiae of WT-SN animals including fibronectin 1 (Fn1, 4.0), Postn (2.4-fold), Itgb4 (2.0-fold), Tnn (1.8-fold), fibulin 2 (Fbln2, 3.0-fold), laminin-alpha 4 (Lama4, 2.4-fold), lambin-beta 1, subunit 1 (Lamb1-1, 2.4-fold), glycoprotein 38 (Gp38 (4.0-fold) and thrombospondin 2 (Thbs2, 3.1-fold). Down-regulated genes in this group include histone cluster4, h4 (Hist4h4, −3.6-fold), Tgfb1 (− 2.0-fold), interleukin 16 (Il16, − 1.7-fold)) interleukin receptor, type 2 (Il1r2, − 1.6-fold), interleukin 17 receptor E (Il17re, − 1.6-fold) and interleukin 1 receptor accessory protein (Il1rap, − 2.2-fold)."

So removing the sciatic nerve is bad or neutral for height growth.

More genes altered by estrogen deficiency(24 hours after loading):

"The down-regulated genes included a number involved in adhesion (Lim and senescent cell antigen-like domains 1 (Lims1, − 2.1-fold), Cd44 (− 1.9-fold)[CD-44 degrades hyaluronic acid so it is bad for height growth and a good thing that it is downregulated], Spp1 (− 1.7-fold)), Wnt signaling pathways (frizzled 5 (Fzd5, − 1.6-fold), casein kinase II beta subunit (Csnk1g, − 1.8-fold) and Csnk2b (− 1.5-fold)) and matrix mineralization extracellular matrix protein 2 (Ecm2, − 1.6-fold) and Mmp13 (− 1.5-fold)[MMP-13 can trigger terminal differentiation of chondrocytes so it's downregulation is very good for height growth]"

So loading under Estrogen deficiency is good for height growth.  Estrogen deficiency increase Estrogen Receptor Alpha levels as well so there may be no net change in Estrogen response.

The Estrogen deficient[<-But not totally eliminated] mice had the best gene expression changes for growth but not that the Estrogen Receptor Alpha mice did not express as many "grow tall" genes this could be that Estrogen Receptor-Alpha lowers circulating levels of Estrogen and thus with no ER-alpha more estrogen runs free or Estrogen Receptor-Alpha helps height growth and estrogen deficiency increases ER-alpha levels therefore increasing height growth genes.  You'd have to knockout all the Estrogen receptors to be sure what the cause of the effects are.

"In the experiments reported here, a single 30-s period of dynamic loading engendering strains of physiological magnitude was sufficient to initiate a cascade of events culminating days later in measurable increase in bone formation."<-30 seconds is the loading performed by LSJL, once per day.