Cistanche Research: Pharmacologic Epigenetic Modulators Of Alkaline Phosphatase in Chronic Kidney Disease Part 2

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EPIGENETIC REGULATION OF ALKALINE PHOSPHATASE

The term Epigenotype was coined in 1942 by Waddington who concluded that between genotype and phenotype lies a whole complex of development processes'[84]. The modern definition of epigenetics includes modifications of DNA and associated proteins, not involving changes to the underlying DNA sequence, that are influenced by the environment and maintained during cell division that cause stable changes in gene expression [85&]. The main epigenetic factors are DNA methylation, posttranslational changes of histones, and higher-order chromatin structure. Post-translational modifications of histones impact chromatin structure, accessibility, and recruitment of transcription machinery to dictate whether genes are switched on or off. These dynamic modifications orchestrate cellular responses to environmental, developmental, or metabolic stimuli through modification of the transcriptome. However, epigenetics can underlie dysregulated gene expression in disease states including cancer [86] and pathological inflammatory processes [87]. Enzymes or proteins that generate or interact with epigenetic alterations can be classified as writers, erasers, or readers, depending on whether they add, remove, or recognize a posttranslational modification (Fig. 3).

FIGURE 3. Chromatin is comprised of DNA and proteins that generate a compact structure critical for the packaging and stability of eukaryotic chromosomes. The primary protein components are histones, around which the DNA is wound to form a nucleosome. Epigenetics involves covalent modifications to chromatin that does not affect the underlying DNA sequence. Covalent modifications to chromatin impact both chromatin structure and recruitment of transcription complexes that, in effect, switch genes on or off. These dynamic epigenetic modifications are carried out by adding (writing) and removing (erasing) posttranslational modifications, followed by ‘reading’, which dictates gene expression and eventual phenotypic response.

Histone acetylation

Histone acetylation is associated with open chromatin structure, accessibility for transcription factor binding, and active transcription [88& ]. Histone of acetylation impacts alkaline phosphatase expression. Histone deacetylase inhibitors (HDACi) increase chromatin acetylation. In vitro, HDACi-induced expression of alkaline phosphatase-L promoted osteogenic differentiation of human mesenchymal stem cells [89]. Mechanistically, histone acetylation has been associated with the regulation of bone morphogenetic proteins, WNT signaling, and RUNX2 induction [90]. Whether acetylation directly impacts promoters of alkaline phosphatase-L expression is an area of ongoing research.

DNA methylation studies have demonstrated that the alkaline phosphatase-L promoter A1E is highly methylated [91]. Delgado-Calle et al. [92], demonstrated that DNA methylation has an important role in the modulation of alkaline phosphatase expression in human osteoblast-like cells. They showed an inverse relationship between the methylation status of a CpG island extending from -579 to þ836 bp of the alkaline phosphatase gene including the promoter region, which implies that epigenetic regulation by DNA demethylation strongly enhances alkaline phosphatase expression and activity [92]. In VSMC, both phosphate and hydroxyapatite nanocrystals modulate DNA methylation, which results in increased alkaline phosphatase activity and the induction of an osteoblast-like phenotype [93,94]

Cistanche can avoid chronic kidney disease.

MicroRNAs

Long noncoding RNAs and microRNAs are also key epigenetic factors that are involved in posttranscriptional gene regulation [77,95& ]. The microRNAs are small non-coding single-stranded RNA molecules, approximately 18–25 nucleotides, that inhibit protein synthesis by binding to the 30- untranslated region of mRNA to block protein translation and/or modulate mRNA stability. It has been estimated, through computational predictions, that more than 50% of all human protein-coding genes are potentially regulated by microRNAs [96]. Bone-regulating microRNAs play a key role in osteogenic differentiation and signaling pathways involved in osteogenesis [77,95&,97,98]. The key transcription factors Runx2 and Osx are downregulated by numerous microRNAs in pluripotent mesenchymal cells to suppress the bone phenotype in nonosseous cells and tissues [77,99].

Some microRNAs have been found to suppress and promote distinct signaling pathways related to osteogenic differentiation [95&,100& ]. Reduced mRNA expression for collagen I, alkaline phosphatase, and osteocalcin has been found while overexpressing miR- 375, thus suggesting that miR-375 can suppress osteogenic differentiation by targeting Runx2 [101]. Overexpression of miR-133a-5p has also been reported to inhibit alkaline phosphatase expression and mineralization through targeting Runx2 [102]. Li et al. [103], demonstrated that miR-216a promoted osteoblast differentiation and enhanced bone formation.

PHARMACOLOGIC EPIGENETIC INTERVENTIONS TARGETING ALKALINE PHOSPHATASE

MicroRNAs

Given the ubiquitous expression of alkaline phosphatase, its central role in biomineralization, and the high incidence of vascular calcification in patients with chronic kidney disease, it is reasonable to explore pharmacologic epigenetic modulation of alkaline phosphatase as a potential therapeutic measure aimed at the prevention of cardiovascular complications in chronic kidney disease [9& ]. Recent evidence indicates that microRNAs are deregulated in chronic kidney disease–mineral and bone disorders [104]. Experimental studies support the concept that microRNAs are potential targets to ameliorate vascular calcification [100& ]. According to the miRBase version 22, sequences of 2656 mature human microRNAs have been cataloged so far [105]. Hence, it is a challenging task to include most of the microRNAs that have been investigated over the years in this review. However, recent data demonstrate that phosphate-induced aortic calcification trigger miRNA modulation by upregulating miR-200c, miR-155, and miR-322, whereas miR-708 and miR-331 were downregulated [106& ]. Other microRNAs that are involved in vascular calcification, thus potential treatment targets, are miR-29a/b, miR-30d/e, miR-125b, miR-135a, miR-143, miR-145, miR-204, miR223 and miR-762 [107]. Most of these microRNAs target the two main transcription factors Runx2 and Osx that influence TNalkaline phosphatase activity and biomineralization. Undoubtedly, microRNAs have a key role in regulating the progression of vascular calcification; however, the high abundance of microRNAs requires extended large-scale epigenome-wide studies to fully exploit the potential of epigenetic regulation by microRNAs for novel therapeutic approaches to ameliorate vascular calcification.

Bromodomain and extra terminal inhibition Bromodomain and extra terminal (BET) proteins BRD2, BRD3, BRD4, and BRDT are chromatin readers that not only bind acetylated lysine on histone tails and transcription factors via bromodomains 1 and 2 but also recruit transcriptional machinery to regulate gene expression [108]. BET inhibitors (BETi) block the interaction of BET proteins with acetylated histones or transcription factors to impact the expression of target genes [88& ]. Apabetalone is an orally available BETi in clinical development for the treatment of cardiovascular disease. It preferentially binds bromodomain 2 in BET proteins (Fig. 4), which distinguishes it from pan-BETi that target bromodomains 1 and 2 with equal affinity [109]. In clinical trials, apabetalone treatment reduced major adverse cardiac events (MACE) in patients with cardiovascular disease and was associated with 44% relative risk reduction on top of standard of care [110& ]. The reduction of MACE by alphabet alone was associated with a reduction of serum alkaline phosphatase, independent of traditional cardiovascular risk factors and inflammation [111& ]. Studies showed this drug concurrently modulated factors that promote atherosclerotic plaque stabilization and MACE reduction. HDL cholesterol increased [110&,112], while the complement cascade, acute phase reaction, and mediators of vascular inflammation were suppressed [113,114].

FIGURE 4. Chromatin acetylation is an epigenetic modification associated with open chromatin structure and active transcription. Bromodomain and extra terminal proteins are ‘chromatin readers’ that bind acetylated lysine on histones or transcription factors via two tandem bromodomains 1 and 2 and recruit transcriptional machinery (e.g. positive transcription elongation factor and RNA polymerase II) to drive expression of bromodomain and extra terminal sensitive genes. Apabetalone is an orally available small-molecule inhibitor of bromodomain and extra terminal bromodomains that causes bromodomain and extra terminal protein release from chromatin and, as a consequence, downregulation of bromodomain and extra terminal sensitive gene transcription. Apabetalone preferentially targets bromodomain 2 (represented by yellow halo), a characteristic that differentiates it from pan-bromodomain and extra terminal inhibitors that bind bromodomains 1 and 2 with equal affinity.

In chronic kidney disease patients with a history of cardiovascular disease, alphabet alone treatment improved kidney function and reduced circulating levels of alkaline phosphatase [115]. Mechanistically, apabetalone downregulated alkaline phosphatase expression in primary human hepatocytes and VSMC [116& ], and as a consequence, reduced TNalkaline phosphatase protein levels and enzymatic activity. Small molecule inhibitors of alkaline phosphatase have been evaluated as a therapeutic for vascular calcification [14], however, apabetalone may be the first clinical-stage molecule to modify alkaline phosphatase production. In vitro, apabetalone opposed calcification of VSMCs cultured in osteogenic conditions through an epigenetic mechanism involving BRD4 that suppressed induction of procalcific genes, including RUNX2 and alkaline phosphatase [116& ].

A single dose of apabetalone in chronic kidney disease stage 4–5 patients rapidly resulted in a reduction of numerous inflammatory cytokines, including IL-6 [2]. In the same study, proteomic profiling of more than 1300 plasma proteins predicted several immune and inflammatory pathways were activated in patients with impaired kidney function, including nuclear factor-kB (NF-kB), IL-6, or bone morphogenetic protein signaling. These canonical pathways were downregulated with one dose of apabetalone, which would favorably impact the progression of renal impairment and associated vascular calcification.

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Bromodomain and extra terminal inhibition in metabolic bone disorders: implication for renal osteodystrophy

Distinct preclinical models of metabolic bone diseases have demonstrated that BETi does not diminish bone structure or mechanical properties, and may instead increase bone volume and restore mechanical strength [117–120]. These studies show that the beneficial effects of BETi on bone disorders stem from anti-inflammatory effects, as well as epigenetic modulation of key factors in bone remodeling, including TN-alkaline phosphatase. N-methyl pyrrolidone (NMP) is a U.S. Food and Drug Administration-approved drug excipient identified as a bioactive BETi [121]. Studies with NMP in preclinical models of bone degeneration have positioned BETi as a pharmacologic strategy for the prevention or treatment of bone diseases characterized by excessive bone resorption. Numerous studies have demonstrated BETi suppresses inflammatory responses mediated by TNFa and NF-kB [3,122–124]. NMP promoted the growth of mineralized bone that was blocked by TNFa and recovered TNFa-inhibited expression of essential osteoblastic genes, including alkaline phosphatase, RUNX2, and SP7/Osterix [125]. In addition, NMP promoted bone regeneration by enhancing BMP2 signaling in osteoblasts [126] and inhibited osteoclast differentiation to attenuate bone resorption induced by receptor activator of NF-kB ligand [127]. NMP was shown to increase osteoblast viability during hypoxia and countered hypoxia-mediated downregulation of key genes involved in mineralization, including alkaline phosphatase [128]. Mechanistically, the NMP treatment was protective in maintaining osteoblast differentiation during hypoxia in part by inhibiting NF-kB signaling. NMP preserved bone mineral density and quality of bones in ovariectomized rats [121], essentially ameliorating estrogen depletion-induced osteoporosis. Results were verified in similar studies using N, N-dimethylacetamide [127], or the more potent BETi JQ1, where treatment reversed bone loss induced by estrogen deficiency [117]. These data imply that BETi therapy can increase bone mass and improve bone turnover in inflammatory bone disorders and potentially in chronic kidney disease.

CONCLUSION

Circulating alkaline phosphatase is a robust and independent risk marker for cardiovascular disease and mortality in the general population and chronic kidney disease. The ubiquitous expression of alkaline phosphatase and its involvement in several pathophysiologic processes associated with cardiovascular disease, bone disease, chronic kidney disease progression, and cognitive dysfunction renders it suitable for multifactorial epigenetic interventions. Positive results from clinical studies with the novel BETi apabetalone implicate a role for alkaline phosphatase as a possible novel cardiovascular treatment target. Experimental studies with additional BET is and microRNAs suggest a wider therapeutic potential for epigenetic modulation of alkaline phosphatase. Further research is required to definitively establish alkaline phosphatase as a clinical treatment target level and to elucidate the effect of lowering serum alkaline phosphatase towards specific targets levels on clinical outcomes.

Acknowledgments

P.M. is supported by ALF grants Region O¨stergo¨tland, Sweden. K.K.-Z. is supported by the NIDDK grants R01- DK095668 and K24-DK091419 as well as philanthropic grants from Mr. Harold Simmons, Mr. Louis Chang, Dr. Joseph Lee, and AVEO.

Financial support and sponsorship

None.

Conflicts of interest

M.H. is a member of the renal clinical advisory board of Resverlogix Inc. and an employee of Diaverum Sweden, AB. He has received consultancy and speaker honoraria from Resverlogix and Amgen. D.G. and E.K. are employees of Resverlogix. K.K.-Z. is a member of the renal clinical advisory board of Resverlogix. P.M. has no conflict of interest related to this article.

REFERENCES AND RECOMMENDED READING

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