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).

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