Wednesday, November 25, 2009

Growth Taller via Autophagy?

Autophagy may be a way to resist aging related degenerations.  Autophagy, unlike apoptosis, allows for recycling of cellular components so if all the hypertrophic cells in the growth plate underwent autophagy over apoptosis it may be possible to grow taller indefinitely.

Regulation of autophagy in human and murine cartilage: hypoxia-inducible factor 2 suppresses chondrocyte autophagy.

"HIF-2 was expressed abundantly by cells in human and murine articular cartilage and in the cartilage of mineralizing vertebrae from neonatal mice. Protein levels were reduced in articular cartilage from older mice, in end-plate cartilage from mice, and in chondrocytes from human osteoarthritic (OA) cartilage. HIF-2 was robustly expressed in the prehypertrophic cells of mouse growth cartilage. When HIF-2alpha was silenced, the generation of reactive oxygen species was found to be elevated, with a concomitant decrease in catalase and superoxide dismutase activity. Suppression of HIF-2 was associated with decreased Akt-1{pAkt was decreased as well} and mammalian target of rapamycin activities, reduced Bcl-xL expression, and a robust autophagic response, even under nutrient-replete conditions. In these silenced chondrocytes, HIF-1 expression was elevated. Decreased HIF-2 expression was associated with autophagy in OA tissues and aging cartilage samples. The autophagic response of chondrocytes in HIF-2alpha-knockout mouse growth plate showed an elevated autophagic response throughout the plate.
HIF-2 is a potent regulator of autophagy in maturing chondrocytes. HIF-2 acts as a brake on the autophagy-accelerator function of HIF-1."

"The maturing cells in [growth plate] cartilage undergo a series of phenotypic changes which include secretion of a unique sets of proteins into the avascular extracellular matrix, up-regulation of alkaline phosphatase and the release and subsequent mineralization of matrix vesicles. Prior to deletion from the plate, the mature hypertrophic chondrocyte becomes glycolytic and undergoes functional and immunohistochemical changes that are characteristic of autophagy. HIF-1, a transcription factor that responds to the tissue oxemic state, promotes chondrocyte autophagy"

"upregulation of HIF-2 lowers intracellular ROS levels by promoting the activities of the dismutating proteins, catalase and superoxide dismutase. The observation that HIF-2 knockout animals are small suggests that there may be an increased rate of chondrocyte apoptosis that serves to impede normal long bone growth."<-so increased autophagy may not increase height however this may be due to other effects like reduced Akt and MTor.

"When we compared neonatal cartilage in the mineralizing vertebrae with end plate cartilage of older mice a dramatic decrease in HIF-2α expression is seen."

HIF-2 reduces reactive oxygen species.  It's possible that autophagy results in the recycling of everything including things that would reduce height growth.

Oxygen may reduce HIF-2 levels providing the signal for epiphyseal mineralization.

"TOR plays a critical role in autophagy serving as the sensor that integrates metabolic and growth factor signals.  Terminal chondrocytes exhibit autophagic characteristics. Morphologically, the arrested cells contain double membrane vacuoles; there is a loss of membrane structure, limited staining and organelle destruction. Since the life history of the growth plate chondrocyte is very short, even minor disturbances in the metabolic state can result in gross impairment of growth. The induction of the autophagic response, permits the terminally differentiated cells to survive the brief rigors of the harsh local microenvironment."

"in the maturing zone of the growth plate, the central core of cells are most removed from oxygen and may be depleted of growth factors and critical nutrients.  In this hypoxic microenvironment, gradients in nutrients and specific growth factors would be countered to some extent by upregulation of ATP generation through the glycolytic pathway.  The loss of growth factor signaling and nutrient stress would suppress mTOR
and activate autophagy."

The life history of the growth plate chondrocyte is 1-2 days.

"Chondrocytes [express] the energy sensor AMPK-1 and [activation increases] with maturation. Thapsigargin treatment activated AMPK and autophagy in a HIF-1-dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Because of the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1-dependent manner."

"AMPK activity is responsive to hypoxia and hyperosmotic stress. The kinase is sensitive to the AMP/ATP ratio: binding of AMP activates AMPKand induces phosphorylation by the tumor suppressor LKB1. When bound, AMP inhibits it’s dephosphorylation by Protein Phosphatase 2C. AMPK functions as an energy sensor that triggers catabolic pathways that produce ATP, while inhibiting energy-consuming anabolic activities."

"autophagy is stimulated by HIF-1 and that in concert with chondrocyte maturation, there is a profound decrease in ATP and a concomitant rise in AMP, key regulators of AMPK"

Mice growth plate cartilage cells were used.

"epiphyseal chondrocytes express AMPKa1".  AMPKa2 is expressed by osteoblasts but not chondrocytes.

"Thapsigargin is an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPases (SERCA). It raises the cytosolic calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticula (ER; as a result, these stores become depleted, resulting in ER stress and ultimately cell death. In a number of tissues, AMPK is activated both by a decrease in energy charge and by an increase in intracellular calcium flux."

"AMPK regulates the induction of autophagy by modulating the association between Bcl-2 and Beclin-1"

"AMPK suppression leads to a significant activation of Akt-1."

"While AMPKa1 positive cells were seen throughout the growth plate, the most densely stained cells were localized to the pre-hypertrophic and hypertrophic regions of the epiphysis. Remarkably, the location of the maximally positive chondrocytes was similar to that described for both Beclin-1 and LC-3, two major indicators of autophagic vacuole formation"

" when HIF-1 was silenced, AMPK activation was blocked; in contrast, there robust AMPK activation when control chondrocytes were treated with thapsigargin."

"autophagy can serve to delay the onset of apoptosis"

How does Salubrinal affect autophagy?

Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death.

"Autophagy is a conserved, self-digestion process that is activated in response to nutrient limitation but acting also as an alternative death mechanism under certain conditions. It is accompanied by the progressive formation of vesicle structures from autophagosomes to autophagolysosomes orchestrated by autophagy effectors (Atg proteins) and modulators (that is, mTOR-mammalian target of rapamycin as a negative regulator). We demonstrate that cyclosporine A (CsA, an immunophilin/calcineurin inhibitor) induces cell death with some apoptotic features but also accompanied by the appearance of numerous cytoplasmic vacuoles, immunostained for endoplasmic reticulum (ER) and autophagy markers. The induction of ER stress in glioma cells by CsA was evidenced by detection of unfolded protein response activation (phosphorylation of PERK, accumulation of IRE1α) and accumulation of ER stress-associated proteins (BIP and CHOP). Formation of the acidic vesicular organelles, increase of autophagic vacuoles, GFP-LC3 punctation (microtubule-associated protein light chain 3) and LC3-II accumulation upon CsA treatment confirmed activation of autophagy. Decrease of phosphorylation of 4E-BP1, p70S6K1 and its downstream target S6 ribosomal protein demonstrate inhibition of mTOR signaling by CsA. Salubrinal and silencing of PERK and IRE1α partially blocked CsA-induced accumulation of LC3-II{So Salubrinal may inhibit autophagy}. It suggests that ER stress precedes CsA-induced autophagy. Surprisingly, silencing of autophagy effectors ULK1, Atg5 or Atg7 increased the level of active caspases 3, 7 and PARP degradation in CsA-treated cells. CsA induces both apoptosis and autophagy in malignant glioma cells via induction of ER stress and inhibition of mTOR/p70S6K1 pathway, however autophagy is cytoprotective in this context."

"In mammalian cells, PERK (protein kinase-like endoplasmic reticulum kinase), IRE1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6) sense accumulation of unfolded proteins in the ER lumen and activate specific cellular response. PERK phosphorylates the α subunit of the translation initiation factor, eIF2α, which attenuates general protein synthesis and initiates the selective translation of some stress-responsive transcripts. IRE1 is a serine/threonine kinase and also an endoribonuclease, which catalyzes the splicing of the mRNA encoding the transcription factor XBP-1 (X-box binding protein 1). PERK mediates transcriptional activation of LC3 and Atg5 proteins under hypoxic conditions via the transcription factors ATF4 (activating transcription factor 4) and CHOP (C/EBP homologous protein), respectively."

According to Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells., Salubrinal protected from apoptosis but not autophagy.  This may be due to the fact that in this study Salubrinal did not increase ATF4 levels.

"Apoptosis signal-regulating kinase 1 (ASK1) is activated by various types of stress, including, endoplasmic reticulum (ER) stress. ASK1 executes apoptosis and/or autophagy under ER stress. [We used] SH-SY5Y cells overexpressing wild-type (WT) ASK1. We show an important autophagic response and an acceleration of the paraquat (PQ)-induced autophagy with hallmarks as accumulation of autophagic vacuoles, activation of beclin-1, accumulation of LC3 II, p62 degradation, and mammalian target of rapamycin dephosphorylation. Inhibition of autophagy caused an exacerbation of the apoptosis induced by WT ASK1 overexpression{So the question is which would increase height more autophagy or apoptosis?} with or without PQ.  Autophagic response could have a protector role. We found an increase in the phosphorylation of the proteins such as IRE1 and eIF2α in response to both the overexpression of WT ASK1 and pesticide exposure.WT ASK1 overexpression-induced autophagy is an event that occurs in parallel with ER stress activation. The importance of ER stress in the autophagy induced by ASK1 and/or PQ was confirmed with salubrinal, a selective inhibitor of eIF2α dephosphorylation. PQ induces an early ER stress response that is correlated with the activation of autophagy as a protective response, which is accelerated in cells that overexpress WT ASK1."

"Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase family (MAP3K) that activates the c-jun N-terminal kinase (JNK) and p38 pathways by directly phosphorylating, and thereby activating, their respective MAP2Ks (also called mitogen activated kinase kinase [MKK]), MKK4(SEK1)/MKK7, and MKK3/MKK6"

"preincubation with [salubrinal] rescued cells from the apoptotic event (release of cytochrome c from mitochondria into the cytosol, caspase-3 activation, and nuclear fragmentation) induced by exposure to PQ, the overexpression of WT ASK1, and the combination of both"

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