International Journal of Molecular Sciences Article Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells Pyotr A. Tyurin-Kuzmin * , Maxim N. Karagyaur, Konstantin Yu. Kulebyakin, Daniyar T. Dyikanov, Vadim I. Chechekhin, Anastasiya M. Ivanova , Mariya N. Skryabina, Mikhail S. Arbatskiy, Veronika Yu. Sysoeva, Natalia I. Kalinina and Vsevolod A. Tkachuk Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Lomonosov Moscow State University, 119991 Moscow, Russia; [email protected] (M.N.K.); [email protected] (K.Y.K.); [email protected] (D.T.D.); [email protected] (V.I.C.); [email protected] (A.M.I.); [email protected] (M.N.S.); [email protected] (M.S.A.); [email protected] (V.Y.S.); [email protected] (N.I.K.); [email protected] (V.A.T.) * Correspondence: [email protected] Received: 30 May 2020; Accepted: 17 June 2020; Published: 22 June 2020 Abstract: Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks. Keywords: multipotent stromal cells; mesenchymal stem/stromal cells; protein kinase A; functional heterogeneity; single cell analysis; intracellular signaling; PKA-Spark biosensor 1. Introduction Multipotent stromal cells (MSC) play a pivotal role in maintenance of tissue integrity and regeneration, especially in skeletal, connective, and epithelial tissues [1,2]. Despite similar morphology and immunophenotype, these cells demonstrate remarkable functional heterogeneity in terms of differentiation abilities and clonogenic capacities. Thus, recent findings obtained using single-cell RNAseq technologies identified about 10 distinct MSC subpopulations with different differentiation and secretion abilities [3,4]. MSCs are regulated by a number of endocrine and paracrine signals, many of which activate G-protein coupled receptors (GPCRs), such as acetylcholine [5], adenosine [6], angiotensin II [7], angiotensin 1-7 [7], ATP [8], dopamine, GABA [9], glutamate [10], histamine [11], noradrenaline [12], serotonin [13], and sphingosine-1-phosphate [14]. Previously, using single-cell signaling analysis of Ca2+-dependent intracellular signaling in living cells, we demonstrated that MSCs functional heterogeneity could be due to different ability to initiate Gq/calcium-dependent cascades in response to hormones, such as noradrenaline and angiotensin II [7,10]. Moreover, Ca2+-dependent functional activity of MSC is regulated by cyclic AMP (cAMP)/protein kinase A (PKA)-dependent signaling activated by β-adrenoceptors [15]. cAMP/PKA signaling pathway also regulates pivotal functions of MSC, such as adipogenic differentiation [16] or regulation of resident stem cells [17]. Int. J. Mol. Sci. 2020, 21, 4442; doi:10.3390/ijms21124442 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4442 2 of 14 However, the activation of cAMP/PKA pathways in response to hormones in living MSCs was not analyzed yet. This was due to the lack of a convenient and reliable research tool for single-cell registration of cAMP and/or PKA activation in living cells in real time. Recently, a genetically-encoded biosensor of PKA activity was developed [18]. This biosensor, named PKA-Spark, consists of polypeptide forming homo-oligomeric coiled-coil, consensus PKA substrate sequence or specific phosphopeptide-binding domain, and green fluorescent protein (GFP) as a readout. PKA-Spark evenly distributes through the cytoplasm and precipitates after PKA-dependent phosphorylation of PKA substrate motif. As a result, it forms intensively fluorescent droplets or aggregates. These droplets are accessible to protein phosphatases; thus, their forming is reversible [18]. In this study, we explored the activation of Gs/cAMP/PKA signaling in response to several hormones in living MSCs at the single cell level using PKA-Spark biosensor. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks. 2. Results 2.1. MSCs Subpopulations Demonstrate Distinct PKA Activation Cultured MSCs uniformly expressed PKA as we demonstrated using immunofluorescent staining of the catalytic subunit of PKA (Figure1a). Since PKA-Spark biosensor was not used before for analysis of signaling pathways in MSCs, we first examined how cells responded to adenylyl cyclase activator forskolin. Apparently, forskolin induced the formation of SPARK fluorescent aggregates in 48.2 4.7% ± of cells (Figure1b,c). These data indicated that almost half of cultured MSCs are not able to activate PKA despite its presence. Direct PKA activation by cell-permeable analogue of cAMP, 6-Bnz-cAMP induced the formation of fluorescent GFP droplets in 91.2 2.8% of MSC (Figure1b,c), which is ± consistent with its uniform expression demonstrated by immunofluorescence. Therefore, these data indicate that about half of MSCs lack the activity of adenylyl cyclase. Mammalian cells express 10 isoforms of adenylyl cyclase numbered from 1 to 10. To evaluate the expression pattern of adenylyl cyclases in MSC population, we processed the data of single cell RNAseq (scRNAseq) analysis of adipose-derived MSC. The raw data of scRNAseq analysis of both human and mouse adipose-derived MSC are openly available [3,4,19]. Figure1d shows distribution of human MSC expressing mRNA of different isoforms of adenylyl cyclase in scRNAseq subpopulations. Adenylyl cyclase isoforms from 1 to 10 correspond to ADCY1 to ADCY10 genes in humans and Adcy1 to Adcy10 genes in mouse. The last subpopulation in the Figure1d, which is marked as AdCy = 0, shows the cells in which there is no mRNA of all isoforms of these enzymes. The share of MSC lacking the expression of ADCY1 to ADCY10 genes in human MSC was 61.0% (5756 from 9429 cells analyzed in dataset from Reference [19]). Distribution of adenylyl cyclases in mouse adipose derived MSC were similar to human one. The share of MSC lacking the expression of Adcy1 to Adcy10 genes in MSC isolated from mouse subcutaneous adipose tissue were 52.9% (1677 from 3171 cells analyzed in the dataset from Reference [3]) and 75.2% (6151 from 8178 cells analyzed in the dataset from Reference [4]). Thus, more than half of human MSC, as well as mouse MSC, did not express mRNA of any adenylyl cyclase isoforms. 2.2. MSC Demonstrate Heterogeneous Response to the Hormonal Stimuli Next, we explored PKA activation by the most prominent hormonal MSC activators, including adenosine, dopamine, histamine, noradrenaline, and serotonin, which may activate Gs-associated isoforms of receptors to these hormones. Expression of Gs-associated receptors in MSCs was tested using PCR-analysis. Figure2a, as well as Supplementary Figure S1, shows that MSCs express mRNA of adenosine receptors (A2A and A2B), dopamine receptors (DRD1 and DRD5), Int. J. Mol. Sci. 2020, 21, 4442 3 of 14 histamine receptors (HRH2), and serotonin receptors (HTR6, and HTR7). Among the analyzed receptors,Int. J. Mol. we Sci. detected 2020, 21, x allFOR tested PEER REVIEW isoforms, with the exception of HTR4 isoform of the serotonin receptor.3 of 17 The expression of all three isoforms of β-adrenoceptors in MSC we recently reported elsewhere [12]. Figure 1. Multipotent stromal cells (MSC) response to adenylate cyclase and protein kinase A Figure 1. Multipotent stromal cells (MSC) response to adenylate cyclase and protein kinase A (PKA) (PKA)activation. activation. (a) Immunofluorescent (a) Immunofluorescent staining stainingof the catalytic of the subunit catalytic of subunitprotein kinase of protein A in MSC. kinase Scale A in µ MSC.bar Scale100 µm. bar (b 100) Sharem. of (b the) Share cells ofresponding the cells respondingto adenylate tocyclase adenylate activator cyclase forskolin activator (10−6 forskolinM) and 6 4 (10−cell-permeableM) and cell-permeable direct activator direct of protein activator kinase of protein A 6-Bnz-cyclic kinase A AMP 6-Bnz-cyclic (10−4 M). AMPResults (10 are− M).presented Results are presented as mean SE, n = 17–24 in 4 independent experiments on the material of 3 different as mean ± SE, n = 17–24± in 4 independent experiments on the material of 3 different donors. (c) donors.Representative (c) Representative field of view field of viewthe cells of the respon cellsding responding to the sequential to the sequential adding addingof forskolin of forskolin and and6-Bnz-cAMP. 6-Bnz-cAMP. Yellow Yellow arrow arrow marks marks the the cell cell responded responded to the to theforskolin forskolin added. added. Blue Bluearrows arrows show showthe thecells cells responded responded to to 6-Bnz-cAMP 6-Bnz-cAMP added. added. Scale Scale bar bar 100 100 µm.µm. (d ()d Expression) Expression of ofmRNA mRNA of ofdifferent different isoformsisoforms of adenylylof adenylyl cyclase cyclase in humanin human MSC MSC population. population. Raw Raw data data of scRNAseqof scRNAseq analysis analysis of MSCof MSC were downloadedwere downloaded from Reference from Reference [19]. [19]. 2.2. MSC Demonstrate Heterogeneous Response to the Hormonal Stimuli Int. J. Mol. Sci. 2020, 21, 4442 4 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 17 Figure 2. MSC express functionally active receptors activating PKA-dependent signaling. (a) PCR Figure 2.