Study showing natural repair of teeth by dental pulp stem cells is dependent on AMPK activation: Connection between Stem cells, Progeria, and HIV-1

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

A recently published and highly publicized study in the journal Scientific Reports in January of 2017 demonstrated that topical administration of a class of drugs known as glycogen synthase kinase (GSK-3) inhibitors led to the mobilization of resident mesenchymal stem cells in the tooth pulp that had been exposed via the drilling of holes in mice molars [1]. GSK-3 inhibitor-induced stem cell mobilization promoted a natural process of reparative dentin (also spelled dentine) formation that completely restored dentin, leading the authors to conclude that stimulation of endogenous stem cells may represent a novel approach to clinical tooth restoration. Interestingly, activation of the master metabolic regulator AMPK has been shown to promote differentiation of embryonic, adult, and cancer stem cells and several GSK-3 inhibitors, including two used in the Scientific Reports study to restore dentin, have also been shown to activate AMPK in vitro and in vivo by inducing a cellular stress response. Adult (e.g. mesenchymal stem cells) and cancer stem cells exhibit quiescence (i.e. a state of dormancy) that is similar to both T cells infected with latent/dormant HIV-1 and premature senescence (i.e. cells cease to divide) that characterizes cells from the accelerated aging disease Hutchinson-Gilford progeria syndrome (HGPS). Because AMPK activation is critical for stem cell differentiation and T cell activation (and thus latent HIV-1 reactivation) and because several compounds that activate AMPK in vivo alleviate accelerated aging defects and promote proliferation in HGPS cells, stem cell-induced natural restoration of dentin, cancer stem cell differentiation, latent HIV-1 reactivation, and restoration of proliferation in HGPS cells are linked via AMPK activation.

The exposure of inner soft pulp tissue following infection (e.g. caries) or trauma leads to a natural repair process characterized by the mobilization and differentiation of resident mesenchymal stem cells into odontoblast-like cells that secrete reparative dentin, a vital mineral component of teeth that is situated beneath the enamel and forms the bulk of the tooth [1]. Although the formation of a thin dentin bridge covers and seals the tooth pulp from infection, reparative dentin secretion and formation is insufficient to repair large lesions and dentin loss due to dental procedures such as carie removal, necessitating the deposition of artificial mineral aggregates to replace lost dentin and fill the tooth [1].

After demonstrating that Wnt/β-cat signaling and the target gene Axin 2 is upregulated after tooth damage and may thus promote restoration of lost dentin, the authors chose three small molecule GSK-3 inhibitors, BIO, CHIR99021, and Tideglusib to stimulate reparative dentin formation (GSK-3 inhibitors have been shown to promote Wnt/β-cat signaling) [1]. All three inhibitors increased mRNA expression of Axin 2 in 17IA4 mouse dental pulp cells, with a four-fold greater induction of Axin 2 by BIO compared to CHIR99021 and Tideglusib, which showed similar levels of induction. Axin 2 induction in vivo was also tested by drilling 0.13 mm holes in mouse maxillary first molars to expose the pulp, soaking a collagen sponge with the inhibitors and inserting the sponge into the holes, and covering the sponge with a cement cap to protect the tooth [1]. 24 hours after treatment and removal of the teeth, Axin 2 expression was 3-fold higher in inhibitor-treated pulp cells compared to controls (i.e. untreated teeth, mineral aggregate only, or collagen sponge only), indicating that GSK-3 inhibitors enhanced Wnt/β-cat signaling.

Most importantly, the authors next determined if the GSK-3 inhibitors induced the formation of reparative dentin. After maxillary molars were drilled, GSK-3 inhibitor-soaked sponges were inserted and the teeth removed after 4-6 weeks. Treatment with BIO, CHIR99021, and Tideglusib led to statistically increased mineralization compared to controls, with GSK-3 inhibitor-induced mineralization 1.7 times greater than mineral aggregate treatment and 2 times greater than treatment with sponge alone [1]. Additionally, subsequent analysis of each molar revealed that GSK-3 inhibitors induced more reparative dentin at the injury site than collagen sponges alone or mineral aggregates, the secreted dentin filled the entire injury site, and the dental pulp remained vital compared to control cells consisting only of a cap or exposed pulp with no cap, indicating that BIO, CHIR99021, and Tideglusib promoted reparative dentin formation via inducing mesenchymal stem cell mobilization and differentiation into odontoblast-like cells [1].

Interestingly, the induction of cellular stress, mediated by an increase in the AMP/ATP ratio, intracellular calcium (Ca2+) increases, or an increase in the levels of reactive oxygen species (ROS) have been shown to activate the master metabolic regulator AMPK and promote the differentiation of embryonic, adult, and cancer stem cells [2-6]. As AMPK activation has also been shown to play a critical role in enhancing embryonic, adult, and cancer stem cell differentiation, it would be expected that the GSK-3 inhibitors used to restore reparative dentin would do so via AMPK-induced stimulation and differentiation of mesenchymal stem cells into odontoblast-like cells [7-9]. Indeed, AMPK activation has previously been shown to promote osteogenic (i.e. bone forming) differentiation of human adipose tissue-derived mesenchymal stem cells [9].

Recent evidence indicates that certain members of the GSK-3 inhibitor class, including BIO and CHIR99021, also activate AMPK by inducing a cellular stress response. Suzuki et al. demonstrated that GSK-3 phosphorylates the α catalytic subunit of AMPK and thus inhibits AMPK kinase activity [10]. However, inhibition of GSK-3 activity by treatment with the GSK-3 inhibitors CHIR99021, BIO, or lithium chloride (LiCl) increased AMPK kinase activity by enhancing phosphorylation of Thr172 of the endogenous AMPK α subunit [10]. Moreover, LiCl also has been shown to increase lifespan in lower model organisms, implicating AMPK as mediator of lifespan extension induced by chemically distinct compounds [11,12]. Weikel et al. also showed that treatment of a primary human aortic endothelial cell (HAEC) model of type 2 diabetes with the GSK-3 inhibitor CHIR99021 increased AMPK activity and attenuated lysosomal dysfunction, an organelle required for autophagy [13]. CHIR99021 also increased AMPK activity and LC3-II protein levels in the mouse aorta, indicating that GSK-3 inhibition promotes AMPK activation and autophagy induction in vivo [13]. Inhibition of GSK-3 in prostate cancer PC-3 cells by the GSK-3 inhibitors TDZD8, Tideglusib, and TWS119 also significantly decreased the levels of ATP, a well known trigger for AMPK activation [14]. TDZD8 was subsequently shown to dramatically induce AMPK activation as well as phosphorylation and activation of ULK1, a key mediator of autophagy induction [14]. TWS119 has also been shown to increase the number of CD4+ and CD8+ T memory stem cells, a stem cell subset that plays a critical role in mounting effective and long-lasting immunological responses to viruses and cancer cells [15]. Rapamycin, a macrolide that extends lifespan in several organisms and also induces autophagy, activates AMPK in vivo in normal mice and also increases the number of CD4+ and CD8+ T memory stem cells, again implicating AMPK activation as a central node promoting stem cell maintenance and differentiation, lifespan extension, and pathogen elimination [15,16].

Furthermore, T cells that are latently-infected with HIV-1 are analogous to both quiescent adult stem cells and HGPS cells that are nearing premature senescence due to the dominant negative effects of the toxic protein progerin. Because AMPK is critical for T cell activation, stem cell differentiation, and the promotion of lifespan extension, it would be expected that chemically distinct compounds that enhance latent HIV-1 reactivation would also induce stem cell differentiation and “activation” of HGPS cells, alleviating accelerated aging defects and increasing proliferative lifespan [7-9,17,18]. Indeed, the AMPK activators metformin and bryostatin-1 have been shown to promote latent HIV-1 reactivation in a monocytic (THP-89) cell line in an AMPK-dependent manner [19]. Metformin has also been shown to promote the differentiation of human stem-like glioma-initiating cells into non-tumorigenic cells in an AMPK-dependent manner, promote osteoblastic differentiation of mesenchymal stem cells, and alleviate accelerated aging defects in HGPS cells [20-22]. Retinoic acid and/or its derivatives has also been shown to activate AMPK, reduce stem cell-like features in pancreatic cancer cells, enhance latent HIV-1 reactivation, and significantly improve cellular proliferation rates and alleviate accelerated aging defects in HGPS cells [23-26].

Because an increase in ROS, intracellular Ca2+ levels, or an AMP/ATP ratio increase promotes stem cell differentiation, the recent findings that the activated form of AMPK is localized across the entire acrosome in human sperm indicates that the acrosome reaction, a prerequisite for the creation of all human life outside of clinical setting, may also be connected to stem cell differentiation, latent HIV-1 reactivation, and alleviation of accelerated aging defects in HGPS cells [27]. Indeed, both ROS and the calcium ionophore A23187 induces the acrosome reaction in human sperm and promotes latent HIV-1 reactivation [28-30]. 1,25-dihydroxy vitamin D3 (vitamin D) also induces the acrosome reaction in human sperm and significantly improves accelerated aging defects in patient-derived HGPS cells [31,32]. Moreover, the rapamycin analog temsirolimus was recently shown to improve accelerated aging defects in HGPS cells [33]. However, temsirolimus induced an increase in ROS and superoxide levels within the 1st hour and a transient decrease in oxygen consumption and cellular proliferation rates, providing compelling evidence that AMPK activators including rapamycin alleviate accelerated aging defects in HGPS cells by inducing a cellular stress response [33].

Lastly, AMPK activation is also critical for oocyte meiotic resumption and maturation, processes that are critical for efficient oocyte activation, a prerequisite for the creation of all human life [34]. As I originally hypothesized in 2016, AMPK is also likely essential for oocyte activation [35]. Indeed, the calcium ionophore A23187, which activates AMPK, has been extensively used to induce human oocyte activation during intracytoplasmic sperm injection (ICSI) procedures, producing normal healthy children and also promotes cancer stem cell differentiation [36-38]. Additionally, puromycin, a protein synthesis inhibitor that activates AMPK, has also been shown to induce cancer stem cell differentiation and activate human oocytes [39-41].

In conclusion, the evidence presented in the Scientific Reports study showing that GSK-3 inhibitors promote tooth repair by inducing mesenchymal stem cell differentiation implicates AMPK activation as central regulator in odontoblast-like cell formation. Two of the GSK-3 inhibitors used in that study, CHIR99021 and BIO, activate AMPK in vitro and in vivo, indicating that cellular stress-induced AMPK activation underlies the therapeutic efficacy of GSK-3 inhibitors. AMPK activation thus represents a central node that connects stem cell differentiation with T cell activation-induced latent HIV-1 reactivation, alleviation of accelerated aging defects in HGPS cells, and the creation of all human life (via oocyte activation and the acrosome reaction in sperm).

https://www.linkedin.com/pulse/study-showing-natural-repair-teeth-dental-pulp-stem-cells-finley?trk=hp-feed-article-title-share

References

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Metformin and exercise shown to promote telomere transcription and integrity via AMPK activation: Connection between Progeria, aging, & HIV-1 latency

"Hutchinson-Gilford Progeria Syndrome" by The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. Scaffidi P, Gordon L, Misteli T; https://commons.wikimedia.org/wiki/File:HIV-budding-Color.jpg#/media/File:HIV-budding-Color.jpg. "HIV-budding-Color" by Photo Credit: C. Goldsmith. Content Providers: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Metformin and exercise shown to promote telomere transcription and integrity via AMPK activation: Connection between Progeria, aging, & HIV-1 latency

A recently published study in the journal Science Advances in 2016 strikingly demonstrated that activation of AMPK by the anti-diabetic drug metformin and other pharmacological AMPK activators promoted telomere transcription in vitro, leading to a significant upregulation of telomeric repeat–containing RNA (TERRA), molecules that play a critical role in telomere protection [1]. Cycling endurance exercise, which is associated with AMPK activation, also led to increased TERRA levels in vivo in healthy human subjects. Interestingly, telomere length has been shown to be significantly reduced in cells derived from patients with the accelerated aging disorder Hutchinson-Gilford progeria syndrome (HGPS) and telomere shortening in normal cells that occurs during cellular senescence activates progerin production, a toxic protein that leads to an accelerating aging phenotype in children with HGPS. Metformin has also recently been found to alleviate pathological aging in HGPS cells by reducing the levels of progerin and SRSF1, a gene splicing factor that has been shown to increase progerin production. As increased SRSF1 splicing activity also inhibits reactivation of latent HIV-1 (preventing immune system detection and destruction of the virus), this study provides further substantiation that AMPK activation beneficially links normal aging, accelerated aging, and HIV-1 latency, a hypothesis I first proposed and published in 2015 and 2016 [25,26].

Telomeres are specialized regions of repetitive nucleotide sequences located at the ends of eukaryotic chromosomes that protect chromosomal ends from deterioration [2]. However, continuous cell division leads to telomere shortening, impeding the replenishment of tissues and triggering cellular senescence (i.e. cells cease to divide). Although human telomeres shorten with age, telomeres may be lengthened by the enzyme telomerase [2]. Human telomeres are also protected by a protein complex known as shelterin and by telomeric repeat–containing RNA (TERRA), molecules that that partly associate with telomeres and are transcribed from subtelomeric promoters [1].

In the Science Advances study, Diman et al. initially demonstrated binding of nuclear respiratory factor 1 (NRF1) to all predicted NRF1 binding sites in subtelomeric sequences using a LB37 non–small cell lung carcinoma cell line. NRF1 is a transcription factor that plays a critical role in modulating the expression of nuclear-encoded genes necessary for mitochondrial respiration as well as mitochondrial DNA transcription and replication [3]. AMPK activation has also been shown to increase NRF1 expression [4]. The authors also subjected 10 healthy volunteers to a cycling endurance exercise for 45 min to determine if AMPK activation is associated with upregulation of TERRA and PGC-1α (peroxisome proliferator–activated receptor γ coactivator 1α). PGC-1a is transcription factor that is activated during exercise or caloric restriction, acts as a transcriptional coactivator for NRF1, and is also upregulated via AMPK activation [1,5]. Indeed, low (50% VO2 peak) and high (75% VO2 peak) intensity exercise led to a significant increase in phosphorylated acetyl–coenzyme A carboxylase (ACC, a marker of AMPK activation), PGC-1α nuclear translocation (indicating its activation), and TERRA induction (186% for high intensity and 131% for low intensity exercise). Post-exercise (2.5 hours) blood lactate levels, which correlated with AMPK activity, were also significantly correlated with TERRA induction, indicating that AMPK activation promotes telomere transcription [1].          

Additionally, knockdown of NRF1 in Huh-7 cells reduced endogenous TERRA levels by 25 to 45 % whereas NRF1 overexpression stimulated luciferase activity in subtelomeric sequences, indicating that NRF1 plays a role in promoting basal telomere transcription. Furthermore, treatment of Huh-7 cells with phenformin, a biguanide similar to metformin that also activates AMPK, increased ACC phosphorylation, induced nuclear accumulation of PGC-1α , increased TERRA levels (185 to 400% of expression in untreated cells), and also increased subtelomeric luciferase activity, indicating that AMPK activation increases TERRA levels by promoting NRF1 and PGC-1α -mediated telomere transcription [1].  

Most importantly, the authors demonstrated that AMPK activation also induces telomere transcription and TERRA upregulation in healthy non-dividing muscle cells. Fluorescence in situ hybridization (FISH) experiments revealed that myotubes expressed an average of 36 telomeric signals and 38 TERRA foci per nucleus, with an average of 35 TERRA foci colocalizing with telomeres, suggesting that a majority of telomeres may be covered by TERRA [1]. Strikingly, myotube treatment with the AMPK activators metformin, phenformin, or AICAR increased ACC phosphorylation and significantly upregulated TERRA levels by 1.6 to 2.2, providing compelling evidence that pharmacological- or exercise-induced AMPK activation promotes telomere integrity by increasing TERRA levels through induction of telomere transcription [1]. 

As noted above, telomere maintenance has also been shown to play a significant role in the production of progerin, a toxic protein produced in both normal humans and patients diagnosed with HGPS via aberrant alternative splicing of the LMNA gene [6]. Normal lamin A plays a critical role in supporting nuclear architecture and morphology. Lamin A binding to subtelomeric repeats also localizes telomeres to the nuclear periphery and loss of lamin A leads to defects in telomeric heterochromatin, altered nuclear distribution and shortening of telomeres, inefficient processing of dysfunctional telomeres by non-homologus end joining, and increased genomic instability [7]. Indeed, telomere length has been found to be significantly reduced in fibroblasts derived from HGPS patients and a recent study also confirmed that in normal human fibroblasts, progressive telomere damage that occurs during cellular senescence activates progerin production and also leads to extensive changes in alternative splicing of many other genes, highlighting a striking similarity between normal aging and accelerated aging in HGPS patients [6,8].

As the splicing factor SRSF1 has been shown to increase progerin production by promoting the use of a cryptic splice site located in the LMNA gene, metformin was recently shown to significantly reduce the expression of SRSF1 and progerin and also improve the nuclear architecture of HGPS cells, indicating that AMPK activation by metformin beneficially alters gene splicing in HGPS cells by modulating SRSF1, a hypothesis that I first proposed and published in 2014 [9,26]. Also, p32, a splicing-associated protein that is an endogenous inhibitor of SRSF1 and is critical for the maintenance of mitochondrial functionality and oxidative phosphorylation, has also been shown to be essential for rapamycin- or starvation-induced autophagy mediated by ULK1 [10,11]. Because rapamycin, also an AMPK activator in vivo, improves accelerated aging defects in HGPS cells by reducing progerin levels via autophagy induction, AMPK activation likely also beneficially modulates the activity of p32, leading to inhibition of SRSF1 splicing activity and enhancement of mitochondrial functionality [12,13]. Moreover, PGC-1α, which is activated by AMPK and promotes telomere transcription and increased TERRA levels, is downregulated in HGPS cells, leading to significant mitochondrial dysfunction [14]. Methylene blue, which activates AMPK in vivo, was shown to increase PGC-1α levels, induce progerin solubility, and alleviate accelerated aging defects in HGPS cells [14,15].

The inhibition of SRSF1 and the promotion of telomere transcription by metformin via AMPK activation also connects HGPS and telomere integrity with HIV-1 latency. Increased splicing activity of SRSF1 inhibits reactivation of latent HIV-1 residing in infected immune cells, preventing immune system detection and destruction of the virus [16]. Reactivation of latent HIV-1 leads to a reduction in SRSF1 but an increase in p32 activity and bryostatin-1 (a PKC modulator) has been shown to reactivate latent HIV-1 via AMPK activation [16,17]. Interestingly, p32 modulation via AMPK activation may also enhance and stabilize the splicing activities of hnRNPA1, a heteroribonuclear protein that associates with p32, antagonizes the splicing function of SRSF1, prevents splicing of the HIV-1 genome (promoting viral reactivation), and participates in the maintenance and preservation of telomeres [18-21]. hnRNPA1 is also decreased in senescent human fibroblasts and antagonizes cellular senescence and the senescence-associated secretory phenotype (SASP) via increasing SIRT1 expression [22,23]. SIRT1, a histone deacetylase that plays a role in a number of age related diseases and in the extension of lifespan, is also activated by AMPK [5].

The evidence presented in the Science Advances publication further substantiates that AMPK activation represents a central node that connects the therapeutic benefits of chemically distinct compounds in diseases as seemingly dissimilar as HGPS and HIV-1 latency. Indeed, AMPK activators including metformin promote telomere transcription and integrity, decrease the splicing activity of SRSF1 that increases progerin production and prevents latent HIV-1 reactivation, and potentially beneficially modulates the activity of the SRSF1 inhibitor p32 and the ribonucleoprotein hnRNPA1. As AMPK activators (e.g. ionomycin) induce human oocyte activation (giving rise to normal healthy children) and AMPK is localized throughout the entire acrosome in human sperm (likely promoting the acrosome reaction), AMPK activation links normal human aging, Progeria, and HIV-1 latency with the creation of all human life [24-27].

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