Signaling Pathways Associated with Maturation

Phenotypic switching is regulated by growth factors, GPCR agonists, ECM molecules, and other mediators found in the bronchoalveolar lavage (BAL) of patients with asthma or COPD^(133,134). The contractile phenotype induction is attainable after exposure to either TGF-β, insulin, or laminin^(135-138). Also, maturation can be supported by lacking of mitogens; in primary ASMC cultures (see Fig.3C), high cell confluence lead to cell cycle arrest by cell contact^{(82, 86)}. sm-specific protein expression is enhanced by RhoA/Rho Kinase and/or PI3-K activation. RhoA/Rho kinase promotes actin polymerization as a downstream effector of either GPCR- or RTK-associated pathways (see Fig.5). Subsequent phosphorylation events, RhoK→ phospholipase D (PLD) → LIMK-1→ cofilin could be responsible for G-actin polymerization into F-actin⁽¹³⁹⁾. A decrement in globular actin level releases some proteins into the cytosol, like the transcriptional coactivator megakaryocytic acute leukemia/ megakaryoblastic leukemia-1 (MAL/MKL-1), which can be trafficked into the cell nucleus and make a macrocomplex with both myocardin (other coactivator) and SRF, a transcription factor. As a whole, they bind gene promoters to increase sm-specific gene expression^{(140, 141)}. TGF-β can amplify this pathway for hypercontractile phenotype induction. Type-1 and type-2 TGF-β receptor activation induce phosphorylation and nuclear translocation of Smad proteins that bind to SRF, building up a macrocomplex similar to MAL/MKL-1/myocardin/SRF, with subsequent gene expression modifications⁽¹⁴²⁾.

Figure 5. Signaling pathways involve in preserving the ASM in a differentiated state.

The Akt/PI3-Kpathway also affect sm-specific gene expression through the transcription factor FOXO-4^{(143, 144)}. Unphosphorylated FOXO-4 binds myocardin and inhibits its association with SRF. Hence, myocardin is released once PI3-K phosphorylates FOXO-4. However, the reach of this pathway goes far beyond transcriptional regulation. Signaling pathways that converge to ribosomal regulation are needed to complete ASMC maturation, which include effectors such as PI3-K, Akt-1, mammalian target of rapamycin (mTOR), and p70 ribosomal S6 kinase (p70^S6K). Pharmacologic inhibition of PI3-K and mTOR are enough to prevent p70^S6K activation and sm-specific protein accumulation⁽¹⁴³⁾. Moreover, activated mTOR can phosphorylate eIF4 binding protein-1 (4E-BP1), releasing and increasing the eukaryotic initiation factor-4 (eIF4) activity⁽¹⁴⁵⁾, followed by contractile protein accumulation (see Fig. 6B).

Figure 6. Signaling pathways involve in the control of ASM phenotypes. (A) Modulation, (B) Maturation.

Persistence of the c-ASMC phenotype is highly dependent on caveolae membrane system and cav-1 expression (see Fig. 5)⁽¹⁴⁶⁾. These flask-shaped structures are classically considered as special compartments for signaling regulation. Cav-1 is located inside those microdomains, being responsible for their formation and maintenance. Interestingly, cav-1 leads to reduced basal activity and sequestration of several receptors and signal transducers related to synthetic/proliferative phenotype induction, such as: PKC, PDGFR, EGFR, Src, and p21ras⁽¹⁴⁷⁾. Proteins such as α-subunit of G proteins, Rho family members, adenylate cyclase isoforms, 7TM receptors, and others with binding domains to glycosyl- phosphatidylinositol are regulated as well. Caveolae act on behalf of cell adhesion by linking the actin cytoskeleton with the basal lamina, facilitated by laminin-2/α₇β₁ integrin interaction. The triggered downstream pathway activates a guanine nucleotide exchange factor, RhoGEF, leading to Rho Kinase activation⁽¹⁴⁷⁾. For this reason, it is possible that mitogen stimulation is not enough to ignite ASMC modulation and cell division, thus caveolae disassembly would be a strict requirement⁽¹⁴⁸⁾.

Laminin is a trimer that bind integrins and non-integrin receptor subtypes, including the dystrophin-glycoprotein complex (DGC). PI3-K inhibition prevents both ASMC maturation and accumulation of DGC proteins, β- and α-DG⁽¹⁴⁹⁾. Preferential expression of DGC in c-ASMC is related to a tighter regulation of reactions to contractile agonists by caveolae system. There is also evidence suggesting that DGC, through β-DG, influences signal transduction by scaffolding properties and interactions with cav-1⁽¹⁵⁰⁾. Additionally, basal activity and low grade stimulation of some RTKs, such as the insulin receptor (PI3-K pathway) and GPCRs (Rho Kinase pathway), could have a role in contractile phenotype conservation, especially in confluent cells. These observations acquire more relevance considering that the ASM mass is likely composed of c-ASMC under physiological conditions. In the disease-setting, ECM component dissolution by MMPs affect the ASMC attachment, shutting down the Rho kinase activity. Unconstrained ASMCs are more susceptible to paracrine influences. Thus, high levels of cytokines and growth factors, and subsequent anomalous repair due to TGF-β, might match with a continuous and dynamic process of phenotypical modulation (contractile → synthetic/ proliferative → hypercontractile/ fibrotic), explaining the ASM thickening and dysfunction.

Signaling Pathways Associated with Modulation

Transition to a synthetic/proliferative phenotype is enhanced by mitogens such as PDGF, EGF, IGF, fibronectin, collagen-I and -II, bradykinin, GPCR agonists, cigarette smoke extract, lipopolysaccharide (LPS), and reactive oxide species (ROS)^{(151, 152)}. Modulation is quickly reached when in vitro ASMCs are not confluent under mitogenic influences (see Fig. 3B), especially fetal bovine serum (FBS) and fetal calf serum (FCS)^{(86, 122)}. Associated pathways converge to increase c-fos (coactivator) expression, which paradoxically needs a prior SRF activation in order to alter gene expression⁽¹⁴⁴⁾. Nevertheless, this apparent duality could be due to a wide variety of transcriptional coactivators that assertively induce selective gene transcription. Contrasting the SRF-myocardin complex effect, SRF cooperativity with ternary complex factors (TCF) such as Elk-1 affects gene promotors with a CArG (CC(AT)₆GG) sequence, inducing cell proliferation instead of maturation. In this way, c-fos upregulation is key to halt contractile gene expression⁽¹⁵³⁾. Furthermore, this coactivator Elk-1 is phosphorylated and activated by the extracellular signal- regulated kinase (ERK)-1 and -2⁽¹⁵⁴⁾. These kinases along with p38, c-Jun N- terminal kinase (JNK), janus kinases (JAKs), and transcriptional factors, like NFκB and AP-1, could participate in signal delivery to increase synthetic and proliferative activities⁽¹⁵⁴⁾. Protein synthesis associated with modulation is also favored following S6 ribosomal subunit phosphorylation⁽¹³⁸⁾. Increased cytoskeleton metabolism with actin polymerization blockade generates G-actin accumulation that avoids nuclear translocation of MAL/MKL-1. Also, it has been described that in vitro both maturation and modulation are reversible.

ASMC modulation is improved by Th2 cytokines. A mix with TGF-β and leukotriene D4, triggers the expression of 29 transcription factors⁽¹⁵⁵⁾. IL-13 is relevant to control the expression of aroung 300 locus. Its receptor, the IL-13Rα1/IL-4Rα complex, mediates the phosphorylation of STAT-6, triggering MAPKs for phenotypic modulation⁽¹⁵⁶⁾. IL-13 can also affect calcium dynamics by upregulation of sarcolipin, which is a transmembrane protein placed at the sarcoplasmic reticulum (SR) that inhibits the sarco/endoplasmic reticulum Ca²⁺- ATPase (SERCA) activity⁽¹⁵⁷⁾. Expression of calcium regulatory proteins changes with modulation, therefore, a decrease in voltage-dependent calcium channels, ryanodine receptors, and SERCA2 levels translate into a calcium dynamics dominated by wave-like propagations. This kind of flow contributes to MAPK pathway activation. Ca²⁺ waves also affect the conformational stability of cis elements in 5’untranslated regions (UTRs) of mRNAs and interactions between translational components, regulating protein synthesis⁽¹⁵⁸⁾. IL-13 signaling is under control of the type-1 suppressor of cytokine signaling (SOCS-1), a protein with chaperone properties. SOCS1 expression is decreased in asthmatic ASMCs and its inactivation raises synthetic activities when exposure to Th2 cytokines⁽¹⁵⁹⁾. In summary, diverse signaling pathways are responsible for driving phenotypic modulation of ASMCs (see Fig. 6A).

Muscarinic Activation leads to ASMC modulation

ASM thickening is attainable by persistent muscarinic stimulation⁽¹²⁷⁾. Both pathways, G_i/0 coupled M₂ and G_q coupled M₃, could generate the activation of MAPK, Rho- kinase, and PI3-K signaling⁽¹¹⁷⁾. Moreover, shifting from a synthetic-proliferative to a contractile phenotype is accompanied by a decrease in M₂ and a parallel increase in M₃ expression⁽¹⁶⁰⁾. Those observations inquire whether or not cholinergic stimulation may affect phenotypic switching. Accordingly, long-term incubation of rabbit ASMCs with ACh or carbachol (CCh) induced a switch towards s/p-ASMC⁽¹⁶¹⁾. Prolonged treatment of bovine ASM strips with the methacholine also diminished contractile protein expression⁽¹⁶²⁾. Transition to a synthetic-proliferative phenotype is characterized by M₃ downregulation and blunted contractile responsiveness to cholinergic stimulation⁽¹⁶¹⁾. In cited studies, signaling pathways were not evaluated, but considering that cholinergic-induced mitogenesis is related to MAPK activation, it is possible that muscarinic activation allows the nuclear translocation of Elk-1, affecting gene expression linked to the phenotype transition.

Role of non-coding RNAs on Phenotypic Stability

The miRNAs are small noncoding RNAs that have an outstanding participation in gene expression regulation. It makes them excellent candidates to control cell plasticity. Multiple mechanisms are involved in miRNA synthesis and gene regulation, as it was previously described⁽¹⁶³⁾. Shortly, mature miRNA is part of the active RNA- induced silencing complex (RISC) that mediates miRNA/mRNA interaction in a specific fashion. This interaction mostly occurs in the 3’UTR by partial complementarity, thus, miRNAs inhibit elongation during translation, or destabilize mRNA promoting its degradation. In smooth muscle biology, multiple miRNAs regulate cell differentiation and proliferation, under physiological and pathological conditions, especially in vascular smooth muscle, although little is known about ASM (see Table 2)⁽¹⁶⁴⁾.

Around 11 miRNAs are upregulated in cytokine-exposed ASMCs. Particularly, miR-25 is significantly modulated after prolonged OVA-challenge⁽¹⁶⁵⁾. Some cytokines through the ↑miR-25/↓KLF-4 system promote ASMC modulation⁽¹⁶⁶⁾. The transcription factor kruppel-like factor-4 (KLF-4) represses sm-specific gene expression by recruiting histone H4 deacetylase activity to smooth muscle cell genes, thereby blocking SRF association with methylated histones and CArG box chromatin. Next-generation sequencing identified miR-10a as the most abundant miRNA expressed in primary human ASMCs, accounting for more than 20% of all small RNAs. miR-10a directly suppresses PI3KCA expression and its overexpression reduces ASMC proliferation⁽¹⁶⁷⁾. TNF-α-induced expression of miR-708 in asthmatic ASMCs is greater than in non-asthmatic. miR-708 decreased JNK, MAPK and Akt phosphorylation and increased MAPK phosphatase-1 (MKP-1) and phosphatase and tension homolog (PTEN)expression. It constitutes a negative feedback for TNF-α signaling downregulation⁽¹⁶⁸⁾. miR-133a levels were decreased in human ASMCs, along with upregulated RhoA expression during AHR. Those findings were replicated after treatment with IL-13⁽¹⁶⁹⁾. Sonic hedgehog signaling blocks miR-206 expression to increase the release of BDNF by ASMCs, coordinating branch innervation⁽⁵²⁾.