Lupine Publishers | LOJ Pharmacology & Clinical Research
Introduction
In molecular biology, the
established central dogma is the widely accepted framework of genetic
information flow. This process begins with the DNA transcription from the
nucleus of the cell into linear, single-stranded messenger RNA (mRNA). Ribosomes
then read the mRNA strand following transportation to the cytoplasm, and
translation of the code into single amino acids, which are added sequentially
to the growing peptide. Once the peptide is fully synthesized, both the mRNA
strand and the peptide are released from the ribosome [1]. Although this
pathway has been established universally as the only way for protein synthesis,
only 1.5% - 2.5% of the human genome codes for proteins [2]. The remainder of
the genome produces noncoding RNAs (ncRNAs). These ncRNAs can be further
classified into various subtypes; of note are miRNAs, small nucleolar RNAs
(snoRNAs), long noncoding RNA (lncRNA), and circular RNA (circRNA) [3].
Although ncRNAs have always been thought to have no role in protein synthesis, many
previously unknown indirect functions of ncRNA have been elucidated within the
past five years. They have been shown to be involved in many settings such as
macrophage activation during an immune response, abnormal expression in
diabetic wounds, retinal diseases, and even endometrial physiology and disease
states such as endometriosis [4-7]. Perhaps the most groundbreaking findings in
the realm of ncRNAs have been their association with various human cancers [8].
Most research has been focused on exploring the epigenetics and
post-transcriptional activity of ncRNA, which allows these molecules to exert
regulatory effects on the expression of the genome [9]. By directly binding to
mRNA strands, miRNAs are able to regulate their ability to eventually lead to
protein synthesis [10]. It has been demonstrated that this process is extremely
precise, allowing targeting of mRNA with high specificity. Interestingly, the
miRNA family has identical 5’ regions with the 3’ region differentiating these
molecules and their specificities [11]. Although protein translation and miRNAs
have conventionally been thought to have such an indirect relationship, our
recent discoveries suggest a much more direct engagement between the two. We
have shown that the sequences in certain pri-miRNAs, more specifically
pri-miR-200a and pri-miR-200b, contain ORFs that are able to be recognized by
ribosomes and subsequently translated directly into protein products,
miPEP-200a and miPEP-200b. Even more intriguing is the potential role of these
pri-miRNA-derived peptides (miPEPs) in cancer repression [12-15].
Recently we have studied the
expression of miPEP-200b in mammalian cells by raising polyclonal antibodies to
miPEP-200b. Our results demonstrate that miPEP-200b is expressed in both breast
and prostate cells (Figure 1). These results establish the presence of
pri-miRNA-encoded proteins in mammalian cells. Many studies have been conducted
on the implications of the miR- 200 family in cancer development and
repression; however, its association with cardiovascular pathology has not been
a central area of focus. Because the miR-200 family has been shown to affect specific
cellular pathways in various conditions, it would not be farfetched to
speculate on its implications in cardiovascular diseases known to have
abnormalities in the same cellular pathways. It was found that a single gene
variant for the miR-200 family could cause increased protein kinase A (PKA)
activity, with subsequent thrombocyte activation and ensuing atherosclerosis
[16]. In addition, PKA has been shown to act as a promoter of human smooth
muscle cell (HSMC) calcification [17]. These processes are known to be the
initial steps to the eventual development of vascular calcification [18].
Previously, we have shown that pri-miRNAs of 200a and 200b code for miPEP-200a
and miPEP-200b respectively and these miPEPs function like miR-200a and
miR-200b suggesting that Nature preserved this duplication of functions in case
of a failure in any one of their functions. As such, we hypothesize that in the
same way that miR-200b plays a role in decreasing PKA activity in
atherosclerotic processes, the peptide miPEP-200b (encoded by pri-miR-200b) may
also act as an inhibitor of PKA-induced HSMC calcification (Figure 2).
Furthermore, targeted miPEP-200b, miRNA-200b, and PKA inhibitor therapy may
lead to a substantial decrease in ISH development in older patients, with an
ensuing decrease in the incidence and prevalence of diastolic heart failure
secondary to longstanding hypertension. In this article, we will discuss the
various components that provide evidence for these potential relationships and
their sequelae.
Mirna and Mipep Interaction
Although there is a significant
amount of information to be discovered regarding the functions of miPEPs,
certain recent important findings allow us to predict potential activities of
miPEPs in relation to miRNA. Of note is the study by Lauressergues et al. which
showed that in plant cells, miPEPs behave as positive feedback on miRNA
production [19]. It is possible that this relationship will also be shown in
human physiology under controlled experiments in the future. This relationship
is quite interesting, given the scarcity of positive feedback mechanisms
observed in nature. In addition, various experiments in previous years
involving miRNAs that attributed their observed findings to the effects of
miRNAs alone may need to be revisited. As an example, it has been demonstrated
that miRNA-200a and miRNA-200b are involved in the suppression of the
epithelial-to-mesenchymal transition (ETM) in certain cancer types [20].
Although miRNA itself was originally thought to be the mediator of this
observation, our recent findings demonstrate that the observed ETM suppression
may be a result of miRNA alone, miPEP alone or a combination of both miRNA and
miPEP activity [12]. Allowing this scenario to serve as a framework (Figure 3)
in many other cellular pathways, one can imagine the vast number of cellular
processes that may, in fact, have a different mechanism than what was
previously proposed [21,22].
PKA and Aortic Calcification
The vasculature anatomy consists of
3 main layers: tunica intima, tunica media, and tunica adventitia. In the
setting of aortic wall calcification, the media layer is involved. The main
component of this layer is HSMC, which aids in constriction and dilation of
vessels by contracting and relaxing, respectively (Figure 4). However, during
the pathological process of medial aortic calcification, these cells begin to
behave as osteoblasts, evident by upregulation of several markers associated
with bone synthesis, including greater alkaline phosphatase activity [23].
Although there may be several mechanisms involved in this process, one
explanation is the over-activity of PKA. PKA is an enzyme found in a multitude
of cell types and tissues in the body. The enzyme is activated via cyclic AMP,
and following activation, it is able to phosphorylate its substrates [24].
Abnormal PKA activity, therefore, causes various downstream effects. In
particular, increased PKA activity may have important pathological implications
in vascular calcification. There seems to be a potential mechanism involving
PKA-induced elevation of parathyroid hormone (PTH). This may then induce medial
aortic calcification [25]. with one experiment involving an in vivo model of
rats with elevated PTH levels causing substantial calcification of the aorta
[26]. In another study, it was shown that inorganic phosphate (Pi), which acts
as a stimulator of PKA, led to HMSC calcification. In this experiment, HSMC
were treated with Pi, calcium levels were measured, and subsequently, PKA
inhibitors were added, then calcium levels were remeasured. It was demonstrated
that inhibition of PKA activity by utilizing siRNA led to a greater than 50%
decrease in HSMC calcium levels [27]. Although this study utilized siRNA as the
inhibitor of PKA, Magenta et al. suggest that an increased PKA activity may be
observed due to a single nucleotide polymorphism (SNP) where a thymidine
nucleotide was substituted by cytosine in genes coding for the miR- 200family
[16]. This may suggest that the miR-200family plays a significant role in
moderating PKA activity.
Aortic Calcification, Hypertension,
and Heart Failure
A major sequela of vascular,
specifically arterial, calcification is the development of hypertensive
disease. As a result of the calcific process involving the media, the arterial
system (including the aorta) becomes stiffened and loses its ability to dilate
and decrease systemic vascular resistance. The resulting hemodynamic state is
such that an isolated elevation in systolic blood pressure is observed, leading
to both ISH and concomitant increased pulse pressure (difference between
systolic and diastolic blood pressure). Although ISH has a strong association with
medial calcification, its relationship with intimal atherosclerosis has not
been elucidated to a significant degree [28]. With sustained, chronic
hypertension, cardiac ventricular muscle cells undergo hypertrophy as a means
to compensate for this increased afterload. Although this mechanism is
compensatory, it is not without pathological consequence, with severe left
ventricular hypertrophy (LVH) eventually leading to diastolic heart failure
[29].
Discussion
Among the numerous functionalities
that miRNA-200b has been shown to have [30], its suggested role in
downregulating PKA activity may be an important player in preventing medial
aortic calcification. This can be inferred as increased PKA activity has been
associated with the deposition of bone-like material in the aortic medial
layer. Although many cellular pathways may potentially be involved, one
proposed mechanism is the PKA-induced increase in PTH levels. Interestingly,
PTH is generally thought to be involved in bone resorption. However, it appears
to have the opposite effect leading to calcification in the vasculature.
Following calcification of the aorta and other arteries, the stiffening of
these vessels leads to ISH. With longstanding ISH, the myocardium becomes
hypertrophied as a compensatory mechanism with the eventual development of
diastolic heart failure. This proposed sequence is presented in Figure 1.
Furthermore, miPEP may prove to be paramount in maintaining high miRNA
activity, given the positive feedback that miPEP exerts on miRNA; although this
observation has only been made in plant cells, the existence of both miPEP and
miRNA in humans could provide the same relationship. No such studies have been
published on the potential regulatory role of miPEP on miRNA in human
physiology due to the relatively recent discovery of miPEP in our previous
study [12]. A very recent study showed that miRNA-8 and miPEP-8 act to produce
the same end result, regardless of their exact mechanisms of action, in
Drosophila [31]. These findings are quite interesting, simulating many other
protective mechanisms seen in nature, such as genetic redundancy [32]. By
providing two mechanisms of accomplishing the same end goal, one mechanism
serves as the main actor, and the other serves as the back-up in case of an event
that renders one of them nonfunctional. As such, even in the case of a
knock-out polymorphism of miRNA-200b, miPEP-200b can independently regulate the
function of PKA by interfering with its regulatory subunit [16] and decreasing
vascular calcification.
Conclusion
If our proposed mechanism of disease
progression in medial aortic calcification in the context of miRNA-200b and
miPEP- 200b is shown to hold true, it will provide targets for therapeutic
interventions. PKA antagonists, miRNA-200b, and miPEP-200b could be utilized
with the end goal of decreasing PKA activity and decreasing vascular
calcification with a subsequent decrease in the incidence and prevalence of
both ISH as well as diastolic heart failure. Furthermore, there is potential
for miRNA-200b and miPEP-200b levels to serve as prognostic factors for these
diseases. There is reason to suggest this, as multiple studies have shown the
promising potential of using miR-200 family levels as a strong prognostic
marker for disease severity; low levels of the miR200 family have shown to be
accurate predictors of a worse prognosis in pancreatic, lung, gastric, and
bladder [33-36]. Although there is much to be discovered in this new arena
involving miPEP, these findings provide an excellent starting point for future
experimental designs, arming scientists with a new outlook on cellular pathways
that were previously thought to behave differently.
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