Structure and Roles of Phospholipase C (PLC), Phosphatidylinositol 4,5-bisphosphate (PIP₂), and Inositol 1,4,5-trisphosphate (IP₃) in Metabolism and Disease: A Systematic Review Open Access

The phospholipase C (PLC)-phosphatidylinositol 4,5-bisphosphate (PIP₂)-inositol 1,4,5‐trisphosphate (IP₃) (PLC-PIP₂-IP₃) pathway is central to cellular communication, serving as a critical mechanism for converting external stimuli into intracellular responses. Its importance lies in its ability to regulate fundamental processes, such as calcium signaling, gene expression, and cell growth, which are essential for maintaining cellular homeostasis.^[1–3] It also plays a pivotal role in functions such as neural transmission, immune responses, and cardiovascular function.^[4–6] Dysregulation of this cascade could be linked to various pathological conditions, including cancer, metabolic disorders, and neurodegenerative diseases, highlighting its clinical significance.^[7–9] Furthermore, the diversity of PLC isoforms and its impact on the distribution and modulation of PIP₂ and IP₃ highlight the pathway’s complexity and flexibility, which is not fully understood.^[1,2] A comprehensive review of the mechanisms and factors is crucial for identifying innovative therapeutic strategies to modulate the pathway’s functionality in the context of diseases while ensuring its critical physiological functions.

This systematic review paper aimed to elucidate the basic structure and metabolic mechanism of PLC, PIP₂, and IP₃ in the human system, delineating their roles specifically for different PLC isoforms and discussing future therapeutic potentials. Furthermore, it explored the interdependent relationship among these components and their downstream impacts. PLC, PIP₂, and IP₃ play pivotal roles in executing various cellular processes across mammalian cells. Upon activation by diverse stimuli in eukaryotic cells, PLC selectively catalyzes the hydrolysis of PIP₂ to generate IP₃ and diacylglycerol (DAG).

In this systematic review, we employed a curated set of keywords for our search, used in different combinations and order: ([phospholipase c] OR [phospholipase c isoyzmes]) AND [Phosphatidylinositol 4,5-bisphosphate]) AND [Inositol trisphosphate]). We conducted an exhaustive search within the databases of PubMed, Web of Science, and Scopus to address the following pivotal research questions: What are the therapeutic potentials and associated challenges of targeting the PLC-PIP₂-IP₃ signaling pathway while also considering the roles of its isoforms and their tissue-specific distributions? The search would focus on the isozymes of PLC, specifically variations of the enzyme. Initially, we identified and saved 618 full-text articles in Endnote, limited to 10 years, during the search process that was reviewed in December 2024. The filter and screening process is detailed in Figure 1. Ultimately, our review incorporated 42 articles that met our criteria and scope; 29 other sources were retrieved when reported in the retrieved articles as citations and additional relevant sources when necessary.

Examples of the pathway’s function are to regulate systems, such as the parasympathetic nervous system, and its functionality heavily relies on the progression facilitated by IP₃ and Ca²⁺ signaling.^[10] The continuous activation and inactivation of the parasympathetic nervous system hinges on the activation of G-protein coupled receptors (GPCR). The excitatory GPCR Gq receptors can stimulate PLC and protein kinase C (PKC), operating in distinct regulatory pathways to modulate Ca²⁺ influx and signaling.^[11] Likewise, the sympathetic nervous system also employs Ca²⁺ to initiate bodily functions geared toward physically demanding activities facilitated through nicotinic receptors that are triggered by acetylcholine. ^[12]

A more comprehensive depiction of the PLC-PIP₂-IP₃ signaling cascade is depicted in Figure 2, expressing the behavior of the signaling pathway between the plasma membrane and the endoplasmic reticulum (ER).^[13] The PLC-PIP₂-IP₃ signaling cascade is a pivotal regulatory mechanism governing calcium signaling and influx. PLC catalyzes the hydrolysis of PIP₂ by PLC, resulting in the formation of diacylglycerol (DAG) and IP₃.^[14] IP₃ triggers the release of Ca²⁺ from the ER and undergoes dephosphorylation to form inositol, which is recycled for PI synthesis. DAG activates PKC in the presence of calcium ions, phosphorylating downstream effectors such as AKT for various cellular functions. Additionally, DAG is phosphorylated into phosphatidic acid (PA), transported into the ER via class IIa PI/PA transfer proteins, and within the ER, PA is converted into cytidine diphosphate DAG (CDP-DAG) by CDP-diacylglycerol synthase (CDS) enzymes, a rate-limiting step in the PIP₂ signaling cascade. CDP-DAG is further converted into PI by phosphatidylinositol synthase (PIS) and transported back to the plasma membrane through transport proteins to regenerate PIP₂.

However, this cycle is not a closed loop, as intermediates can transit to different cells, activating diverse downstream processes. Newly synthesized molecules, like PIs, can enter the PIP₂ cycle.^[15] Moreover, this signaling cascade significantly influences the broader network of inositol metabolism, regulating phosphatidylinositol phosphates and inositol phosphate within the larger cellular signaling framework.^[16] Ultimately, each cascade component has various roles and functions, which will be further summarized in the following sections.

The PLC family encompasses 16 distinct members categorized into the following seven classes: β, γ, δ, ε, η, ζ, and XD, differentiated by splice variants as isozymes.^[1,2] Of note, the XD family is regarded as atypical compared with the other classical members based on its structure, where substrate specificity and activation mechanisms are yet to be confirmed.^[2,3] These members comprise four PLCβ (β1–β4), two PLCγ (γ1, γ2) three PLCδ (δ1, δ3, δ4), one PLCε, two PLCη (η1, η2), one PLCζ, and 3 XD (XD1–XD3).^[3,17] PLC exhibits significant protein structures, such as PH domains, EF-hand motifs, X and Y domains, C2 domains, and PDZ domains, each contributing to distinct catalytic functions.^[1,18] This enzyme regulates cellular PIP₂ levels by localizing within or outside lipid rafts in the plasma membrane and catalyzing phosphatidylinositol hydrolysis reactions.^[1]

Within a mammalian context, PLC cleaves the phospho-ester bond in phosphatidylinositol, generating DAG and an inositol compound phosphorylated at position 1 and other positions within the inositol ring.^[19] The enzyme has a preferential affinity for PIP₂ as a substrate over other potential substrates like PIP and PI.^[1,6] Although all isozymes catalyze the same reaction, their diversity allows for differential regulation of catalytic activity.^[3] Specifically in osteoclasts, PLCγ1 and PLCγ2 have separate and irreplaceable roles in cell differentiation, specifically suppressing differentiation through downregulating CSF-1, β-catenin, and cyclin D1 levels, compared with upregulating NFATc1 for osteoclast differentiation, respectively.^[20] Moreover, the translocation of PLC serves as a crucial regulatory mechanism, where the nuclear localization of PLCβ plays an essential role in myoblast differentiation, while its translocation into cytosolic regions inhibits differentiation.^[21,22]

PLC enzymes play distinct but overlapping roles in various cellular functions and signaling pathways throughout the body, as described in Table 1, with various splice variants and regulatory cofactors. PLCβ1 and β3 exhibit broad expression in various tissue and cell types, whereas PLCβ2 and β4 are, respectively, limited in hematopoietic and neuronal tissues.^[6] Although PLCβ isoforms distribute across different cells and tissues, most contain only one or two measurable isoforms.^[23] Despite differences in enzyme expression among isoforms, all four PLCβ isoforms can be activated by Gα subunits of the Gq class (Gαq).^[6] Additionally, these isoforms respond to various activators, including the βγ subunit of Gα_i.o G protein, Rac GTPases, and Cdc42, particular in the case of PLCβ2 and β3.^[2,6,24] Moreover, these isoforms exhibit distinct responses to their activators; for instance, PLCβ2 is the least responsive to Gαq among the four isoforms but is highly sensitive to Rac activation.^[25] Conversely, PLCβ3 displays heightened sensitivity to Gβγ.^[26]

The regulation of PLCγ isoforms involves receptors or nonreceptor kinases, while Ras, Rho GTPases, and G protein subunits regulate PLCε.^[2,6] Variances in isoforms serve to offer diverse signaling profiles within the PLC-PIP₂-IP₃ signaling cascade and subsequent downstream signaling.^[3] This diversity potentially contributes to spatial and temporal complexity in cell signaling, signaling amplification, and modulation of enzyme activity timings.^[3] The rate of PIP₂ hydrolysis may rely on the mode of PLC activation, which could vary from minimal or transient hydrolysis for a short duration to sustained hydrolysis. Different modes of PLC activation encompass receptor tyrosine kinases, G-protein coupled receptors, monomeric G proteins, or increased cytosolic Ca²⁺.^[3,27]

PLCβ isoforms play crucial roles in immune regulation. Xiao et al^[6] reported significant antibody production in mice lacking both PLCβ2/3, while Bach et al^[28] observed impaired chemokine-mediated T-lymphocyte migration in PLCβ2/3-deficient mice. Moreover, PLCβ, PLCγ, and PLCδ are implicated in macrophage-mediated inflammatory response.^[29] Some PLC isozymes exhibit multifunctional roles, doubling as GTPase-activation proteins or guanine nucleotide exchange factor function; for instance, PLCβ isoforms can serve as GTPase-activation proteins for Gαq.^[30]

PLCs have emerged as potential players in human cancer development and progression, where specific isoforms have been linked to various cancer types, as described in Table 1: PLCβ in neuroendocrine tumors and hematopoietic malignancies, PLCγ in breast cancer, colon carcinoma, lymphocytic leukemia and angiosarcoma, PLCδ in esophageal squamous cell carcinoma, and PLCε in gastric cancer and colorectal cancer.^[7] However, the regulatory roles determining their pro- or anti-oncogenic effects remain elusive, as different PLC isoforms can take diverse roles in altering cell growth, proliferation, survival, and cell migration—key hallmarks influencing cancer development and progression—by impacting various regulatory pathways’ interconnectivity.^[7] These pathways notably include PI3K/Akt/mTOR, RAS/RAF/MAPK/ERK, and the JAK/STAT pathways.^[7] PLCγ1 has also been implicated in impacting metastasis effects such as cell migration and invasion of cancerous cells.^[31] Last, brain disorders resulting from PLC gene alterations have also been observed, especially point mutations, missense, and nonsense mutations to different PLC isoforms were found to be linked to Alzheimer’s disease, Autism spectrum disorder, epilepsy syndrome, and bipolar disorder.^[8]

PIP2, a negatively charged phospholipid, consists of a glycerol backbone, two fatty acid chains, and an inositol head group carrying two phosphate groups. It serves as a substrate for both PLC and PI3K.^[32] The phospholipid makes up approximately 1% of total cellular phospholipids but acts as a major poly-phosphoinositide.^[33] There are seven isoforms differentiated by the phosphorylation positions at the inositol ring at positions 3, 4, and 5.^[33] The most common isoforms that are also the most studied include PIP₂, PI(3,5)P2, and PI(3,4)P2.^[33]

Primarily situated on the cytosolic side of the plasma membrane, PIP₂ facilitates membrane trafficking of ion channels and transporters, regulating intracellular trafficking.^[34,35] Additionally, PIP₂ has been found observed translocating out of the cytosolic side of the plasma membrane, with ATP-binding cassette transporter A1 facilitating the efflux of PIP₂ to apoA1, allowing transport into the plasma by highdensity lipoprotein reaching its target site through the HDL receptor scavenger receptor BI.^[36]

The PIP₂ found in the nuclei are predominantly synthesized by PIP5K (90%) compared with PIP4K, where PIP₂ would be responsible for regulating cell cycle progression.^[22] Functionally, PIP₂ plays a critical role in regulating actin cytoskeleton dynamics, crucial for cell motility, vesicle trafficking, phagocytosis, endocytosis, exocytosis, and actin cytoskeletal reorganization.^[37,38] Particularly in endocytosis and exocytosis, the accumulation of PIP₂ at engagement sites is vital for these processes.^[39] PIP₂ function is also influenced by cholesterol and various divalent metal ions like Ca²⁺, Mg²⁺, and Zn²⁺.^[40]

The precise levels of PIP₂ and PI(3,4,5)P₃ are tightly regulated by the activity of various kinases and phosphatases, such as PI3K, PTEN, and SHIP.^[41] With PIP₂ representing a master regulator for different cellular functions, its homeostasis is essential to maintain its roles for signal transduction; Nir2 and Nir3 can act as feedback regulators. Both Nir2 and Nir3 are suggested to react to PA, a downstream product of PIP₂ hydrolysis. The subsequent DAG phosphorylation product builds up in the ER, which will translocate PI from the ER to the plasma membrane (PM) to resynthesize PIP₂, with Nir2 and Nir3 under different physiological states.^[42,43] A study by Chang and Liou^[42] proposes that at resting state, low PA levels lead to basal level replenishment of PI into the PM to resynthesize PIP₂, while intense build-up of PA as PIP₂ depletes introduces a more potent translocation movement of PI into the PM from the ER across the ER-PM junctions to resynthesize PIP₂. Apart from using Nir2 and Nir3 and transporters, CDP-diacylglycerol synthase enzymes (CDS1 and CDS2) to convert PA into PI at the ER is also a regulatory mechanism to replenish PIP₂ after the PIs are translocated across the ER-PM Junction by the members of the phosphatidylinositol transport proteins and or Nir2 and Nir3.^[14,44]

PIP₂ can bind to ion channels and ion transporters, acting as a regulation mechanism for activation and inhibition purposes, often through conformation of the protein structure, as described in Figure 3.^[35,45]Figure 3 highlights the regulatory roles of PIP₂ in modulating various ion channels and transporters. For example, X-ray crystallography of the K_ir2.2 channel shows that the tetrameric channel for potassium transport opens as one PIP₂ binds to each of the four channel subunits for viable protein conformation. ^[15,46] Overall, PIP₂ can act as a master regulator for the function of multiple types of ion channels with or without the presence of protein kinase C, including transporters, Ca²⁺-activated K⁺ Channels, voltage-gated K⁺ channels, inward-rectifier K⁺ channels, two-pore domain K⁺ channels, Ca²⁺ release and receptor-operated channels, voltage-gated Ca²⁺ channels, renal epithelial Na⁺ channels, transient receptor potential (TRP) channels, and chloride channels.^[32,35,45] Mutagenic studies on basic, hydrophilic, or hydrophobic amino acids of these ion channels or transporters demonstrate altered protein activity through the modification of protein affinity.^[34] Similarly to the protein conformation induced by K_ir2.2’s quaternary structure, PIP₂ can stabilize the norepinephrine transporter dimers at the plasma membrane to modulate substrate efflux.^[47] However, PIP₂ and the binding specificity of certain ion channels or ion transporters would allow for phosphoinositide with higher degrees of phosphorylation when the affinity for PIP₂ is low or moderate, which can allow for certain redundancy for different ion channel or transporter regulation.^[48]

Another example of PIP2’s regulatory role is its essential binding to TRP melastatin (TRPM)8. Ubiquitously expressed in highly cold-sensitive tissues, these ion channels are functionally employed for obtaining external stimuli signals.^[49,50] These tissues include but are not limited to skin, cornea, teeth, tongue nasal mucosa, and the oral cavity epithelium.^[50] Sensitive to both the presence of cold temperatures (>10°C, <28°C) and cooling compounds, the multimodal channel receptor TRPM8 is regulated by PIP₂ and primarily expressed in the sensory and dorsal root ganglion neurons. TRPM8 channels can partake in different physiological functions, including modulating the mechanosensory function of the bladder and its contractibility, regulating prostate ion and protein secretion, and modulating the immune system.^[51]

PIP₂ binding TRPM8 is essential and sensitive to the cooling compounds menthol and icilin in the presence of Ca²⁺ and cold temperature stimulation.^[52,53] However, the cooling agents and cold couples with PIP₂ as conversing sensitizers, where high concentrations of cooling agonists can also increase the channel’s affinity to PIP₂ binding to initiate channel function.^[53,54] Both the cooling agonists and antagonists for TRPM8 bind to the voltage sensor-like domain cavity of the channel. The channel is also regulated by the Ca²⁺ influx function of the channel by activation of Ca²⁺ sensitive PLC and subsequent PIP₂ depletion, while binding of Gαq to TRPM8 can induce inhibition and also influence PIP₂’s depletion.^[54,55] Downregulation of the TRPM8 channel is also achieved by protein kinase C–mediated dephosphorylation, Ca²⁺-dependent protein kinase C isozyme, and ethanol presence.^[56–58] Rapid inhibition of TRPM8 measured currents wasobserved in the presence of both menthol and cold-induced activity, where currents were restored once ethanol was removed from the environment in rate TRPM8.^[58] External environment can also inhibit TRPM8 responses, as the addition of ethanol can also inhibit TRPM8 through interrupting the PIP₂-TRPM8–binding interaction through changes in the surrounding lipid interactions of the channel, leading to reduced binding sensitivity to PIP₂.^[58]

Nuclear PIP₂ can also impact the effectiveness of the cell cycle, where PIP₂ phosphorylation by inositol polyphosphate multikinase to form PI(3,4,5)P₃, which is required to facilitate the double-strand break repair pathway of DNA replication.^[59] Beyond DNA repair mechanisms, PIP₂ is involved in a complex network of cellular mechanisms, including the regulation of histone RNA polymerase activity, chromatin remodeling complexes, splice factors, RNA polymerase, splice factors, and polyadenylation.^[59] From a pathological disease perspective, when PIP₂ synthesis is disrupted, possibly as a result of unsuited ATP:ADP ratios to conduct PIP₂ synthesis, pathological occurrences, such as mitochondrial dysfunction, diseases such as diabetes and cerebral autosomal dominant arteriopathy may occur.^[32] Alzheimer’s may also be observed in humans, having linked PIP₂ with ApoE4 carriers in postmortem brain tissue.^[9]

IP₃, a water-soluble molecule, can enter the cytoplasm and bind to the IP₃ receptors (IP₃R) in the ER to release Ca²⁺ into the cytoplasm. The binding enables a rapid release of Ca²⁺ stored in the ER into the cytoplasm, subsequently inducing DAG activation of protein kinase C to phosphorylate downstream effectors.^[24,38] IP₃Rs are also found in the nuclear envelope.^[60] PLC prefers PIP₂ as its substrate, and different PLC isozymes have distinct tissue distributions and means of regulation.^[1]

The response of the IP₃ receptor to IP₃ obeys the power law, where increased IP₃ levels correspond to increased calcium concentrations.^[24] Increased IP₃ levels release heightened calcium levels, as Hohendanner et al,^[60] demonstrated augmented calcium transients upon cytosolic IP₃ release in atrial myocytes upon field stimulation, triggered by the photolytic detachment of IP₃. This effect was counteracted by 2-APB, an IP₃R blocker, confirming the role of IP₃ in elevating Ca²⁺ signals. IP₃R exhibits three isoforms with varying ratios across different cells, type 2 IP₃R(IP₃R2) being the most prevalent in contractile cardiomyocytes, allowing for modulating responses from IP₃ attachment.^[61] These expressions from IP₃R2s are also driven by IP₃ modulated Ca2+ release and aided by ATP, leading to its own system of self-regulation. Furthermore, Alzayady et al^[62] showed that for the IP₃R tetrameric intracellular ionic channel, all four monomers within the tetramer must be occupied by IP₃ to allow for sufficient calcium ion release upon Ca²⁺ stimulation, as demonstrated through mutating binding sites. IP₃R has a biphasic response to cytosolic Ca²⁺, where low Ca²⁺ concentration induces the ion channel’s activation, while high Ca²⁺ concentration inhibits the ion channel’s opening.^[63] These mechanisms are providing multiple regulatory mechanisms for regulating metabolic functions.

For IP₃ production, a three-dimensional spatial modeling study of PIP₂ signaling in cerebellar Purkinje cell models by Brown et al^[64] in 2008 observed that basal levels of PIP₂ are insufficient to prompt IP₃ production. Yet, Hille et al^[24] proposed that minimal PIP₂ hydrolysis, without net depletion through overexpressing phosphatidylinositol 5-phosphatases, generates enough IP₃ for IP₃ receptor channel activation and calcium release.^[65] These different findings raised the question of the rate-limiting factor for IP₃ to activate IP₃R through the hydrolysis of PIP₂ by PLC and their regulatory mechanisms. Additionally, the duration of IP₃ release can also influence and regulate calcium signals, with prolonged release resulting in distinct calcium responses compared with regular IP₃ release.^[64] Specifically in ventricular cardiomyocytes, significant calcium signal differences were observed between prolonged (100 ms) and regular (2 ms) IP₃ release.^[64]

The calcium release through IP₃ also impacts PLC activity, as all PLC isozymes require calcium binding to induce enzyme activity.^[66] Although all PLCs require calcium, PLCδ is highly sensitive to calcium, where calcium concentrations can act as regulatory mechanisms for PLC activity for PIP₂ hydrolysis.^[66] However, IP₃ induces Ca²⁺ oscillations on a non PLC dependent manner, but through calcium induced calcium release driven at the IP₃R, as oscillations were observed in the presence of the PLC inhibitor U71322.^[67] Furthermore related, ethanol’s presence can also impact the production of IP₃ through PLC activity inhibition, but production is restored once ethanol is metabolized.^[68]

However, compared with ryanodine receptor (RyR)-mediated Ca²⁺ release, another intracellular calcium channel, IP₃R-mediated Ca²⁺ release, has significantly slower kinetics, providing longer lasting Ca²⁺ release (3× longer), but only being 20–25% in signal amplitude.^[69] Also, the amount of Ca²⁺ released by IP₃R is less substantial than RyRs, as RyRs are approximately 100-fold more abundant compared with IP₃Rs, suggesting coupling functions or separate regulatory mechanisms are possible.^[63,70] Overall, the Ca²⁺ released by IP₃R is not the major contributor, but is still a major contributor to calcium release as a regulator and calcium oscillations in cells.

Specific in cardiac pathologies, such as cardiac ischemia, diabetes-induced arrhythmias, or sepsis-induced cardiomyopathy and cardiac hypertrophy respectively implicated for abnormal function of IP₃R1 and IP₃R2, neurons which are hyperactivated without functional closing of receptors can lead to calcium overload in all of the cytosol, the mitochondria and the nucleus of cells.^[71] Similar to PLCs, most cells can express more than one IP₃R isoforms, but often with one isoform acting as the main regulator, as well as having their differences in regulatory sensitivity, adding another imension to the pathway to consider.^[72]

The different PLC isotypes have been observed for their potential in therapeutic outcomes and treatments in different targets for controlling cellular implications. PLC isoforms like the role of PLCβ2 in inducing vascular endothelial growth factor A (VEGF-A)-mediated vascular permeability through PIP₂ and calcium flux, which positively regulates the tissue metabolic homeostasis function post-VEGF stimulation in animals, where VEGF-A is a common stimulus in patients with acute and chronic diseases such as sepsis and cancer.^[73] Likewise, PLCβ3 has been implicated in negatively regulating the same VEGFA-mediated vascular permeability and providing endothelial barrier integrity.^[74] Balancing PLCβ2 and PLCβ3 could help regulate vascular permeability for therapeutic purposes.^[73]

Furthermore, PLCγ1 has been linked with the regulation of the HER2/HER3/PI3K and EGFR/HER2/PLCγ pathways, pathways that dictate migratory and metastatic potential of breast cancer.^[75] Additionally, the use of PLCγ inhibiting U73122 suppressed the expression of proinflammation molecules in orbital adipose tissue from patients with Grave’s orbitopathy, providing potential targets for treatment.^[76] However, it is often denoted that PLCγ, as well as other phospholipases, are “undruggable” due to its involvement in many a large complexion of cellular processes, which can largely be problematic if global or major inhibition of PLCγ can induce devastating side effects.^[31,75] Using specific inhibitors might alter the expression of other factors dramatically, causing unwanted effects in the process. Finding ways to upregulate and downregulate different PLC isotypes to contain the function in specific areas could be challenging, and the complete screening and elucidation on each isotype’s interacting partners could allow for the controlling of clinical outcomes and thus is an important field of research to be able to control PLC function and therapeutic potential.^[75]

Another approach for possible clinical significance is to use PLC functions in conjunction with specific treatment therapies. Rustagi et al suggests that upregulation and related gene therapy methods of open reading frame clones of PLCγ2 improved the outcome of VEGF replenishment therapy through VEGF or PLCγ2 combinatorial gene therapy to treat diabetic-induced limb ischemia, whereas normally the treatment has poor clinical outcomes due to the inability to respond to ligand concentration of VEGF.^[77] Inducing PLCγ1 signaling related to TrKB/BDNF/PLCγ1 signaling was also observed as possibly having clinical significance in acute sleep deprivation settings, where BDNF protein microinjection robustly activated TrkB and PLCγ1 signaling through phosphorylation, which was denoted to have mitigated memory impairment that was tested in short- and long-term memory testing sessions.^[78] As such, there are different approaches to using the potential of PLC isozymes through high-throughput screening studies, as long as the mechanism and signaling pathways are largely elucidated, where unwanted side effects could be minimized, and the therapeutic outcome optimized.

Outside of targeting activation and inhibition of PLCs, miRNAs have also been explored for their roles in the malignancy of pathologies, specifically in breast tumors. Cohort studies of samples from breast cancer patients by Bertagnolo et al^[79] allowed the group to explore the expression of PLCβ2 in early breast cancer cells, which suggested the modulation of PLCβ2 resulting from the downregulation of a tumor suppressor miRNA (miR-146a). Their research suggested that different PLCβ2/miR-146a could characterize the tumor’s malignant potential differently and also be capable of predicting invasive recurrence tendencies in breast cancer patients. It is also an approach to target specific isoforms, although the authors suggest other miRNA might be involved. Such studies encourage scientists to explore different possibilities in harnessing the therapeutic potential of such a complex pathway or provide opportunities to improve current health research.

The PLC-PIP₂-IP₃ signaling pathway can be critical for intracellular communications, regulating calcium signaling, gene expression, and cell growth. The pathway’s dysregulation may be implicated in various diseases, including cancer, metabolic disorders, and neurodegenerative conditions. Although targeting this pathway presents therapeutic potential, challenges arise from the different critical roles of PLC isoforms and their complex regulatory mechanisms, making them difficult targets to drug or alter. Strategies, such as isoforms-specific inhibitors, gene therapy, and microRNA modulation, offer potential solutions.

We found limited comparable data because of the complex, interconnected roles of PLC, PIP₂, and IP₃ as master regulators. Different cofactors, isoforms, isozymes, mutations, distributions, and concentrations in different cells and organs can all impact how the signaling cascade is regulated and what it provides to the body. A single component can impact different parts of the signaling cascade, as presented in Figure 2, and the other interconnected pathways can make the specific components hard to target. Also, as we excluded articles focused on IP₃R and DAG as a focus during our PRISMA literature research as it was outside of our research scope, both having a role in the signaling cascade that can impact their impact on the human body, the review may lack the overarching picture of how the cascade can act and its impact on disease. One such study excluded is the involvement of IP₃R1 in mediating fatty liver.^[80] Ultimately, there is still work to be done as all three components can affect different downstream processes, requiring further complementary research and considerations for clinical significance.

Overall, the complexity of the signaling pathway and its effects across the human body will need refinement of research and data to explore the potential for therapeutic treatment approaches and for the expansion of our understanding of PLC, PIP₂, and IP₃ in their various signaling pathways. Gaps such as the specific roles and mechanisms of the different PLC isoforms in various tissues and their regulatory mechanisms are not fully understood, so the current data may not fully enable the research capabilities for clinical applications. Furthermore, the downstream effects of manipulating these isoforms may lead to unwanted effects if not fully elucidated. Addressing such gaps will further enable our understanding of the complex biological processes and, ultimately, the therapeutic potential for this critical pathway.

Metabolic pathways are used by the human body to control different aspects of cellular functions. In conclusion, the PLC-PIP₂-IP₃ pathway stands as a major regulator of intracellular calcium signaling, orchestrated by diverse PLC enzymes and PIP₂ variations across tissues. The pathway’s modulation affects numerous cellular functions, offering therapeutic potential. However, challenges arise in targeting specific elements due to their involvement in critical processes, demanding further research to unlock clinical applications effectively. A deeper understanding of PLC mechanisms and the modulation of PIP₂ and IP₃ is essential for unlocking their therapeutic potential.

The authors thank Ngai Choi (Shanghai Jiao Tong University, Shanghai, China) for his input, as well as for the additions to the manuscript during the manuscript writing and editing phase.