Thursday, September 20, 2012

Cilium's role in adaptation to LSJL length increase response

LSJL may reduce in it's chondroinitiating ability over time because of the actin cytosekelton or the primary cilium.

The solitary (primary) cilium--a mechanosensory toggle switch in bone and cartilage cells.

"In bone the large number of osteocytes form a vast osteointernet in which the gap junctionally interconnected members are lodged in an extensive lacunocanalicular network. The much smaller number of articular chondrocytes are not interconnected in a chondrointernet [but] are separately lodged in capsules called chondrons. The non-motile solitary (primary) cilia protruding like aerials from osteocytes (as well as osteoblasts) and chondrocytes are switches that when toggled by cyclical pulses of lacunocanalicular fluid or cartilage compression send signals such as Ca(2+) surges into the cell to trigger a cascade of events that include appropriate gene activations to maintain and strengthen bone and cartilage. The chondrocyte cilium with its Ihh(Indian hedgehog)-activated Smo receptor is a key player along with PTHrP in endochondral bone formation."

"The trabecular lattice provides an immense surface for the interaction of bone cells with various hormones and cytokines to release Ca2+ into the blood when needed. Moreover the trabeculae also provide niches (nests) carpeted with retired osteoblasts, called bone-lining cells, that anchor and regulate the long-term hematopoietic stem cells"<-thus osteoblast signaling may play a role in chondroinduction by LSJL.

"Osteocytes must have a flow-metering device that is switched on and off by the pulsing L-C fluid and triggers Ca2+ surges, the expression of the COX-2 gene for PGE2 production and induces the expression of NO synthase that makes NO"

"murine MC3T3 preosteoblasts and MLO-Y4 osteocytes express the Tg737 and Kif3a genes for cilial proteins, express the genes for the PKD1 (PC-1) and TRPP2 (PC-2) signalplex. proteins and indeed have the solitary (primary) cilium protruding from them"<-LSJL does not up- or down-regulate any of these proteins or genes above threshold so maybe the cilia does not play a role in the reduction of adaptation to LSJL over time.

"PKD1 (PC-1) [activates] the P1 promoter of the gene for the Runx2 transcription factor which targets the osteoblast’s genes for α1 (I) procollagen, osteocalcin, osteopontin, and osterix"

"Chondrocytes are encapsulated in fibrillar-walled chondrons which are embedded in an ICM (interchondronal matrix), a hydroelastic collagenous composite. The chondron is distinguished by being selectively enriched with the minor collagen VI{many forms of Col6 are upregulated by LSJL} and water-binding proteoglycans (hyaluoronan and aggrecan)"

"when the cartilage and its chondrons are strongly and cyclically compressed the water associated with the chondron proteoglycans is squeezed through the PCC (pericellular capsule) wall into the much less strained ICM (i.e., the PCC has a smaller compression modulus than the ICM; it takes less force to produce a given amount of PCC strain than ICM strain) leaving the chondron with a higher osmotic pressure because of the compression-concentrated proteoglycans and salts."

"When the compression drops during each cycle, water with chondrocyte nutrients is drawn back into the decompressed chondron because of the high osmotic pressure produced during compression"

"By straining the PCC’s fibrillar meshwork and pushing the PCC wall closer to the cell surface during the flattening of the chondron during each compression cycle the chondrocyte cilium with meshwork fibrils attached to it will be bent "

"The upper part of the cilium is decorated with plaques containing α2β1 and α3β1 integrins which are tethered to the pericellular collagen fibers"

"Cilium generation in murine growth plate chondrocytes can be prevented by disabling the gene for the Kif3a subunit of the kinesin motor that carries the various structural and functional components up along the ciliary microtubules"<-Kif3a is not affected by LSJL above threshold.

"The Ihh receptor complex Smo•Ptc on the cilia of transit-amplying chondrocytes in developing bone is part of the Ihh-PTHrP feedback mechanism that determines the length of the pre-hypertrophic zone and thus the final adult bone length. In adult bones, the cyclical compression produced by various activities sends pulses of fluid through the bones’ extensive L-C steointernet. These pulses toggle the osteocytes’ solitary (primary) cilia which send waves of Ca2+ and Ca2+-mobilizaing IP3 signals through the L-C osteointernet."<-These waves of Ca2+ may initiate chondrogenic differentiation.

Stem cells have cilia as well:

Primary Cilia Mediated Mechanotransduction in Human Mesenchymal Stem Cells.

"The primary cilium is a single sensory cellular extension, which has recently been shown to demonstrate a role in cellular mechanotransduction and MSC lineage commitment. Short periods of mechanical stimulation in the form of oscillatory fluid flow (OFF) is sufficient to enhance osteogenic gene expression and proliferation of human MSCs. Cilium mediates fluid flow mechanotransduction in hMSCs by maintaining OFF-induced increases in osteogenic gene expression and, surprisingly, to limit OFF-induced increases in proliferation. [Primary cilium play a] pro-osteogenic mechanosensory role."

"As little as 1hr of pulsatile fluid flow (PFF) was sufficient to enhance COX2 gene expression in adipose derived hMSCs"<-COX2 gene expression was upregulated in less than an hour of LSJL but that was in rats.  Maybe human cells need longer duration in addition to more load.

"An increase in proliferation of hMSCs [follows] high magnitude, short-term OFF"

"The primary cilium is a singular, immotile microtubule based cellular extension which projects from the apical surface of nearly every cell in the human body"

"Upon HH ligand binding of the Patched1 (PTCH1) receptor, PTCH1 derepresses Smoothened (SMO) which in turn translocates to the tip of the primary cilium activating the Gli transcription factors{LSJL upregulates Gli3}"

Osteogenic differentiation was favored by the media so that is likely why chondrogenic factors were not detected.

"The flow rate was chosen to yield a peak shear stress of 1.0 Pa (28ml/min). For proliferation rate studies the flow rate was adjusted accordingly to yield a peak shear stress of both 1.0 Pa (28ml/min) and 2.0 Pa (56ml/min). For all experiments, cells were exposed to 2 hrs of oscillatory fluid flow."

"Mechanical stimulation did not significantly affect the mRNA expression of RUNX2, OPN, OCLN,
ALKLP, COL1a1 or BSP at any time point."<-this is in contract to LSJL which altered mRNA expression of the majority of those genes.

"higher magnitudes of flow are required to elicit a proliferative response in hMSCs."

When the cilia was inhibited responsed to mechanical stimuli was stunted.  Maybe the cilia play a role in adaptation to stimulis.

"ATP induced proliferation activates PI3K/Akt, mTOR/p70/S6K and ERK1/2 dependent signaling pathways in fibroblasts"

"the application of 30mins of OFF was sufficient to significantly enhance cAMP production in hMSCs"

"adult bone cells which do not possess b1 integrin and/or Focal Adhesion Kinase (FAK) do not respond to fluid shear with an increase in osteogenic gene expression"

"b1 integrins have been shown to localize to the primary cilium, including chondrocytes"

"Polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARgamma, [and Sox9] were measured as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. Primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARgamma mRNA while SOX9 mRNA expression was significantly lower[So lack of cilia reduces chondroinduction but does not inhibit]."

So if cilia does play a role in adaptation then LSJL will still work at inducing chondrogenesis just a reduced level.

"the response of cells to the family of secreted Wnt proteins has also been shown to be regulated by the primary cilium."<-LSJL involves Wnt2.

"The Sox-9 mRNA levels were 0.86 for the polaris transfected group vs. 1.53 for the [group with functional cilia]"

"chondrogenic differentiation may be regulated by additional mechanisms other than signaling cascades involving the primary cilia."<-thus maybe the 2 week decay in adaptation to bone genes to LSJL will be different for chondrogenesis.

Ift88 regulates hedgehog signaling, Sfrp5 expression, and β-catenin activity in post-natal growth plate.

"Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes [results] in disorganized columnar structure and early loss of growth plate. We compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down-regulated. Sfrp5 was up-regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. TSfrp5 is a downstream target of Hh and Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/β-catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of β-catenin. Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling."

"Arl13b{down} [is] a cilia specific protein"

Genes downregulated in primary cilia depleted chondrocytes also downregulated by LSJL:


Intraflagellar transport is essential for endochondral bone formation

"Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. We generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants."

"ectopic chondrocyte-like cells were observed between the perichondrium and diaphysis [in mice lacking Cilia]."

"cells adjacent to the perichondrium resembled chondrocytes rather than osteoblasts. While it is unclear if these cells originated from the perichondrium or the growth plate of the developing bone, they appeared to be continuous with the perichondrium in some sections, suggesting that they may have originated there"

B is where the ectopic chondrocytes are located(where the green arrows highlight).

"the development of ectopic chondrocytes along the diaphysis is not seen in Ihh;Gli3 mutants. Rather, this is characteristic of defects in canonical Wnt signaling."

"mice with conditional loss of β-catenin in the developing skeleton exhibited ectopic chondrocyte differentiation in the perichondrium"

"The ectopic cells in conditional mutants expressed aggrecan and morphologically resembled chondrocytes"

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