RESEARCH ARTICLE

Specific protein 1, c-Abl and ERK1/2 form a regulatory loopJiaoyue Long, Guoning Liao, Yinna Wang and Dale D. Tang*

ABSTRACTThe tyrosine kinase c-Abl participates in the regulation of variouscellular functions including cell proliferation, adhesion, migration,smooth muscle contraction and cancer progression. However,knowledge regarding transcriptional regulation of c-Abl is surprisinglylimited. Sp1 is a foundingmember of the Sp1 transcription factor familythat has been implicated in housekeeping gene expression, tumor cellproliferation and differentiation. Here, we show that knockdown andrescueofSp1affectedgrowth factor-mediatedc-Abl expression incells.c-Abl promoteractivity was alsoaffected bySp1 knockdown. This is thefirst evidence to suggest that Sp1 is an important transcription factor toregulate c-Abl expression. In addition, Sp1 phosphorylation at Thr-453and Thr-739 has been proposed to regulate its activity in Drosophilacells. We unexpectedly found that growth factors did not induce Sp1phosphorylation at these two residues. In contrast, growth factorstimulationupregulatedSp1expression. Intriguingly, inhibitionofERK1and ERK2 (ERK1/2, also known asMAPK3 andMAPK1, respectively)reduced expression of Sp1 and c-Abl. Furthermore, c-Abl knockdowndiminished ERK1/2 phosphorylation and Sp1 expression. Takentogether, these studies suggest that Sp1 can modulate c-Ablexpression at transcription level. Conversely, c-Abl affects ERK1/2activation and Sp1 expression in cells.

KEY WORDS: c-Abl, Sp1, Smooth muscle, Signal transduction,Proliferation

INTRODUCTIONc-Abl (Abelson tyrosine kinase, Abl) is a non-receptor tyrosinekinase that plays an essential role in regulating a variety of cellularfunctions, including cell adhesion, migration, cytokinesis andsmooth muscle contraction (Anfinogenova et al., 2007; Chen andTang, 2014; Cleary et al., 2014; Hu et al., 2005; Wang et al., 2016;Wang, 2014). Furthermore, c-Abl promotes the proliferation ofsmooth muscle cells (Jia et al., 2012; Wang et al., 2013a) andfibroblasts (Mitra et al., 2008) during stimulation with growthfactors. c-Abl has also been implicated in airway smooth musclelayer thickening in asthma (Cleary et al., 2013; Rhee et al., 2011;Tang, 2015). Its mutant form BCR-Abl contributes to thedevelopment of leukemias, such as chronic myeloid leukemia(Wang, 2014).The expression level of c-Abl is associated with various

pathological and physiological conditions including cancer,pregnancy and asthma (Tang, 2018; Tang and Gerlach, 2017;Weigel et al., 2013; Yaba et al., 2011). Although the roles of c-Ablin cell functions and disease pathogenesis have been extensively

investigated, fundamental knowledge regarding the regulation ofits expression is surprisingly limited. Recent studies have suggestedthat c-Abl expression is regulated in part by the microRNA(miR)-203 in T cell lymphoma (Bueno et al., 2008) and smoothmuscle cells (Liao et al., 2015). The mechanisms that regulate c-Abltranscription in human cells are largely unknown.

Specific protein 1 (Sp1) is a founding member of the Sp1transcription factor family, which participates in the regulation ofgene expression. Sp1 was initially identified as a promoter-specificbinding factor that is essential for transcription of the SV40 majorimmediate early (IE) gene (Dynan and Tjian, 1983). For manyyears, Sp1 was thought to be a basal transcription factor thatregulates expression of housekeeping genes, and it has beenimplicated in tumor cell proliferation, differentiation, apoptosisand angiogenesis (Beishline and Azizkhan-Clifford, 2015). TheDNA-binding domains of Sp1 possess three highly homologouszinc fingers, which preferentially bind to the same GC consensussite [5′-GGGCGG-3′] in target promoters and regulate transcriptionof housekeeping genes (Beishline and Azizkhan-Clifford, 2015;Nagaoka et al., 2001). Moreover, Sp1 phosphorylation at Thr-453and Thr-739 has been implicated in modulating its transcriptionalactivity in SL2 Drosophila cells (Milanini-Mongiat et al., 2002).However, how Sp1 is regulated in human cells is still elusive.

The mitogen-activated protein kinase (MAPK) pathway is criticalfor the regulation of various cellular functions including geneexpression (Jia et al., 2012; Wang et al., 2013a; Widmann et al.,1999). Upon stimulation with growth factors within minutes, ERK1and ERK2 (ERK1/2, also known as MAPK3 and MAPK1,respectively) become phosphorylated and activated, whicheventually can regulates gene expression and cell proliferation(Chang and Karin, 2001; Jia et al., 2012; Wang et al., 2013a;Widmann et al., 1999). Interestingly, c-Abl can regulate ERK1/2activation in smooth muscle cells (Jia et al., 2012; Wang et al.,2013a) and fibroblasts (Mitra et al., 2008) upon activation bygrowth factors.

In this study, we used the PROMO online tool (http://alggen.lsi.upc.edu/recerca/menu_recerca.html; for prediction of transcriptionfactor-binding sites) to analyze potential transcription factor-binding sites on c-Abl promoter and found that there are fiveSp1-binding sites on the essential region of the c-Abl promoter.Moreover, we used loss-of-function and rescue approaches toevaluate the role of Sp1 in c-Abl expression. We discovered thatSp1 regulates c-Abl promoter activation, c-Abl expression and cellproliferation. Furthermore, c-Abl conversely controls ERK1/2activation and Sp1 expression.

RESULTSTreatment with PDGF increases c-Abl expression in smoothmuscle cellsBecause c-Abl is one of the major players in regulating smoothmuscle cell proliferation during activation with growth factors (Liaoet al., 2015; Tang, 2015; Tang and Gerlach, 2017; Wang et al.,2013a), we questioned whether PDGF affects c-Abl expression inReceived 6 July 2018; Accepted 27 November 2018

Department of Molecular and Cellular Physiology, Albany Medical College, Albany,NY 12118, USA.

*Author for correspondence (tangd@amc.edu)

D.D.T., 0000-0002-7339-9249

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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs222380. doi:10.1242/jcs.222380

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http://alggen.lsi.upc.edu/recerca/menu_recerca.htmlhttp://alggen.lsi.upc.edu/recerca/menu_recerca.htmlhttp://alggen.lsi.upc.edu/recerca/menu_recerca.htmlmailto:tangd@amc.eduhttp://orcid.org/0000-0002-7339-9249

cells. Human airway smooth muscle (HASM) cells were treatedwith 10 ng/ml PDGF for 24 h, and immunoblot analysis was used toassess c-Abl protein expression. The protein level of c-Abl in PDGF-treated cells was higher than in untreated cells (Fig. 1A). These resultssuggest that treatment with PDGF increases the expression of c-Ablprotein in HASM cells. Because ERK1/2 and AKT (also known asAKT1) are also phosphorylated and activated upon growth factorstimulation (Jia et al., 2012; Liao et al., 2015;Wang et al., 2013a), weevaluated the effects of PDGF stimulation on ERK1/2 and AKTphosphorylation. Phosphorylation levels of ERK1/2 and AKT werehigher in cells treated with PDGF as compared to untreated cells(Fig. 1B,C). Because PDGF treatment increases cell number, weevaluated whether cell density affects PDGF-dependent c-Ablexpression. Cells with different densities were treated with PDGFfor 24 h. c-Abl protein expression was higher in PDGF-treated cellswith 25%, 50% and 75% densities (Fig. 1D).

Exposure to PDGF promotes cell proliferationWe also determined the role of PDGF in cell proliferation. HASMcells were treated with 10 ng/ml PDGF for 24 h. DNA synthesis inthe cells was evaluated using the BrdU incorporation assay (Jiangand Tang, 2015). BrdU incorporation was higher in cells treatedwith PDGF than in control cells (Fig. 1E). In addition, exposure toPDGF increased cell number (Fig. 1F). The results are consistentwith previous results that PDGF promotes cell proliferation(Ambhore et al., 2018; Liao et al., 2015; Wang et al., 2013a).

Sp1 regulates c-Abl expression in cellsWe used the PROMO online tool to analyze potential transcriptionfactor-binding sites and found that there were five Sp1-bindingsites on the essential region of c-Abl promoter (NCBI ReferenceSequence: NG_050744.1) (Fig. 2A). This raised a possibility thatSp1 may regulate c-Abl transcription and expression. HASM cellswere transfected with either control siRNA or Sp1 siRNA for 24 h.They were then cultured in medium containing 0.25% fetal bovineserum (FBS) (to prevent cell death) with or without PDGF foradditional 24 h. Immunoblot analysis verified that Sp1 knockdown(KD) occurred in cells treated with Sp1 siRNA for a total of 48 h(Fig. 2B). Moreover, Sp1 KD did not affect the expression ofproliferating cell nuclear antigen (PCNA) and vimentin (Fig. 2B),two proteins implicated in cell proliferation (Cheng et al., 2016;Cleary et al., 2013). The results indicate that Sp1 siRNA selectivelyinhibits Sp1 expression. More importantly, Sp1 KD inhibited thePDGF-induced c-Abl expression at the protein level by ∼40% andmRNA level by ∼50% (Fig. 2C). Because Sp1 is critical for cellproliferation (Beishline and Azizkhan-Clifford, 2015), completeknockdown of Sp1 can impede cell viability. As such, we used thecell model in which Sp1 had a partial, although still substantial,downregulation, for the following experiments. In addition, Sp1 KDreduced c-Abl expression in cells not treated with PDGF (Fig. 2C).This is not surprising because cells were cultured in medium with alow concentration (0.25%) of FBS. These results suggest that Sp1regulates both basal and PDGF-treated c-Abl expression.

Fig. 1. Treatment with PDGF promotes c-Abl expression and cell proliferation. (A) Human airway smooth muscle (HASM) cells were treated with10 ng/ml PDGF for 24 h or left untreated. Protein expression was evaluated by immunoblot analysis. Data are mean±s.d. (n=4). (B,C) Phosphorylation of ERK1/2(p-ERK1/2; n=7) and AKT (p-AKT; n=4) in cells treated with PDGF for 24 h was evaluated by immunoblot analysis. Data are mean±s.d. (D) Cells with 25%,50% and 75% densities were exposed to PDGF for 24 h or left untreated. The same volume of sample buffer was used to extract proteins. Immunoblot analysisshows that c-Abl expression is higher in PDGF-treated cells with different densities. Data are mean±s.d. (n=3). (E) DNA synthesis in cells treated with orwithout PDGF for 24 h was evaluated with a BrdU incorporation assay. The amount of newly synthesized DNA is higher in PDGF-treated cells. Data aremean±s.d. (n=4). (E) PDGF stimulation increases cell numbers. Data are mean±s.d. (n=6). Ctrl, control. *P

Furthermore, we performed the KD and rescue experiment to testthe specificity of Sp1 siRNA. HASM cells were treated with controlsiRNA, Sp1 siRNA and Sp1 siRNA plus RNAi-resistant Sp1construct for 2 days. Immunoblot analysis verified Sp1 proteinexpression in the Sp1 KD and rescue cells (Fig. 2D). Moreimportantly, rescue of Sp1 in the KD cells restored c-Abl proteinand mRNA expression (Fig. 2D). Taken together, the findingsindicate that Sp-1 promotes c-Abl expression in cells.

Knockdown of Sp1 inhibits cell proliferationHASM cells were treated with either control siRNA or Sp1 siRNAfor 24 h followed by PDGF treatment for an additional 24 h. DNA

synthesis was reduced in cells treated with Sp1 siRNA, as revealedby a BrdU incorporation assay (Fig. 2E). Moreover, Sp1 KDdiminished basal and PDGF-induced cell proliferation (Fig. 2F).Furthermore, Sp1 was located in the nucleus of smooth muscle cellsas evidenced by immunfluorescence analysis (Fig. 2G).

Sp1 regulates the activity of c-Abl promoter in cellsNext, we determined whether PDGF stimulation affects c-Ablpromoter activity. HASM cells were transfected with c-Ablpromoter reporter (Fig. 3A) for 48 h followed by PDGFstimulation for 24 h. Luciferase activity of the c-Abl promoter inthe cells was then determined. PDGF exposure increased c-Abl

Fig. 2. Sp1 regulates expression ofc-Abl mRNA and protein in cells.(A) Schematic diagram of human c-Ablpromoter. TSS, transcription start site.The approximate location of theSp1-binding sites is indicated.(B) HASM cells were transfected witheither 20 nM control or Sp1 siRNA for48 h. Protein levels of Sp1, PCNA andvimentin in these cells were assessedby immunoblot analysis. Data aremean±s.d. (n=4). *P

promoter activity (Fig. 3B), indicating a role for PDGF in activatingthe c-Abl promoter. Because there are five Sp1-binding sites on theessential region of c-Abl promoter (Fig. 2A), we determined the roleof Sp1 in c-Abl promoter activation. HASM cells were co-transfected with the c-Abl promoter reporter, and with eithercontrol siRNA or Sp1 siRNA for 48 h followed by PDGF activationfor 24 h. Basal and PDGF-induced luciferase activity of the reporterin Sp1 KD cells was reduced compared to that in control cells(Fig. 3C). These results indicate that Sp1 has a positive role inregulating c-Abl promoter activity.

Site 1, 4 or 5 affects c-Abl promoter activityTo assess the role of the Sp1-binding sites in c-Abl promoteractivation, we mutated the five GC-rich sites from GGGCGG toGTTCGG. Cells were transfected with wild-type or mutant c-Ablpromoter reporters plus Sp1 construct and cultured for 72 h. Theluciferase activity of the promoter with mutated Site 1, 4 or 5 wassignificantly reduced compared with that of the wild-type promoter(Fig. 3D). In contrast, the luciferase activity of the promoter withmutated Site 2 or 3 was not diminished significantly (Fig. 3D).Furthermore, the luciferase activity of triple mutant (Site 1/4/5) isreduced by 80% (Fig. 3D). The results suggest that Site 1, 4 and 5are important for c-Abl promoter activation and not all binding sites

on the promoter are necessary for Sp1-mediated activation. Theresults are not surprising because not all binding sites are accessibleto transcription factors in certain cell types (Soufi et al., 2015).

PDGF exposure increases the occupancy of Sp1 inendogenous c-Abl promoterTo assess whether PDGF affects the interaction of Sp1 withendogenous c-Abl promoter, smooth muscle cells were treated withPDGF for 24 h. Chromatin immunoprecipitation (ChIP) analysiswas performed using Sp1 antibody. In cells treated with PDGF, theamount of c-Abl promoter fragment precipitated with Sp1 washigher in cells treated with PDGF than in control cells (Fig. 3E).Quantification analysis showed that the Sp1-precipitated DNA wasenhanced in cells treated with PDGF; however, IgG-precipitatedDNA was not altered after PDGF treatment (Fig. 3F).

TreatmentwithPDGFdoesnot increaseSp1phosphorylationat Thr-453 and Thr-739 in smooth muscle cellsERK1/2-mediated Sp1 phosphorylation at Thr-453 and Thr-739 hasbeen shown to promote Sp1 transcriptional activity in SL2Drosophila cells (Milanini-Mongiat et al., 2002). Previous studieshave shown that PDGF exposure activates ERK1/2 in various celltypes including smooth muscle cells (Jia et al., 2012; Wang et al.,

Fig. 3. Role of Sp1 in c-Abl promoteractivity. (A) Schematic illustration of c-Ablpromoter reporter construct as part of adual-reporter system, which uses GaussiaLuciferase (GLuc) as the promoter reporterand SeAP (secreted alkalinephosphatase) as the internal control forsignal normalization (GeneCopoeia).TPS, transcriptional pause site. (B) PDGFtreatment increases luciferase activity ofc-Abl promoter. Data are mean±s.d.(n=4). *P

2013a). Our current results showed that PDGF-induced ERK1/2phosphorylation was increased as early as 5 min after stimulation,and slightly reduced and sustained for 24 h (Fig. 4A,B).Furthermore, we assessed whether PDGF could increase Sp1phosphorylation at the two residues. HASM cells were treated withPDGF for different times, and Sp1 phosphorylation at the residuesin cells was evaluated by immunoblotting. We unexpectedly foundthat the phosphorylation of Sp1 at Thr-453 and Thr-739 was notincreased in cells treated with PDGF (Fig. 4A,C,D).

Exposure to PDGF increases expression of total Sp1 proteinin smooth muscle cellsNext, we sought to explore whether PDGF affects expression oftotal Sp1 protein. Cells were treated with PDGF for different times,followed by immunoblotting. PDGF slightly increased theexpression of total Sp1 protein in cells 4 h after stimulation, butsignificantly enhanced Sp1 expression 24 h after treatment (Fig. 4A,

E). Moreover, PDGF increased the expression of c-Abl protein 24 hafter stimulation (Fig. 4A,F). These results suggest that Sp1transcriptional activity may be regulated by its protein level ratherthan its phosphorylation in smooth muscle cells.

Role of ERK1/2 in Sp1 and c-Abl expressionAs described earlier, ERK1/2 phosphorylation plays an importantrole in the signaling pathways that control gene expression. Wequestioned whether ERK1/2 may regulate Sp1 and c-Abl in cells.First, we found that PDGF exposure increased ERK1/2phosphorylation (Fig. 1B), which is supported by previous studies(Jia et al., 2012; Jiang and Tang, 2015; Liao et al., 2015; Tang,2015). Immunoblot analysis demonstrated that exposure to U0126(an inhibitor of MEK proteins, which are upstream of ERK1/2)inhibited ERK1/2 phosphorylation in smooth muscle cells(Fig. 5A). However, treatment with U0126 did not reduce theamount of total ERK1/2 (Fig. 5A). Therefore, it is unlikely that

Fig. 4. Time course of PDGF-induced protein phosphorylation and expression. (A) Smooth muscle cells were treated with 10 ng/ml PDGF for differenttime points or left untreated. Protein phosphorylation or expression was evaluated by immunoblot analysis. (B–F) Protein phosphorylation or expression instimulated cells normalized to that in corresponding unstimulated cells. The data represent means±s.d. (n=4). *P

U0126 nonspecifically inhibits all cell functions. To determine thefunctional role of ERK1/2, HASM cells were treated with 10 µMU0126 with or without PDGF for 24 h. Treatment with U0126inhibited basal expression of Sp1 and c-Abl at the protein andmRNA level (Fig. 5B–D). More importantly, exposure to U0126attenuated the PDGF-induced upregulation of Sp1 and c-Abl at theprotein and mRNA level (Fig. 5B–D). These results indicate thatERK1/2 has a role in regulating Sp1 and c-Abl expression at thetranscription level in cells. Additionally, exposure to U0126attenuated the basal and the PDGF-induced BrdU incorporation,as well as the amount of basal and PDGF-stimulated cellproliferation (Fig. 5E,F).

c-Abl also regulates expression of Sp1 during PDGFactivationc-Abl has been shown to regulate ERK1/2 activation in various celltypes including smooth muscle cells (Jia et al., 2012; Mitra et al.,2008; Wang et al., 2013a). Because ERK1/2 regulates Sp1expression, we speculated that c-Abl may conversely regulate Sp1expression. We have previously generated and characterized stablec-Abl KD HASM cells (Chen and Tang, 2014; Wang et al., 2013a).

To test the potential effect of c-Abl on Sp1, stable c-Abl KD HASMcells and control cells were treated with PDGF for 24 h and assessedby immunoblotting. Sp1 protein expression in c-Abl KD cells waslower than in control cells (Fig. 6A). Furthermore, we alsoconfirmed that c-Abl KD inhibited basal and PDGF-inducedERK1/2 phosphorylation (Fig. 6B). However, c-Abl KD did notinhibit the expression of total ERK1/2 in cells (Fig. 6B).

DISCUSSIONThe roles of c-Abl in cell functions and disease pathogenesis havebeen extensively characterized. The knowledge regarding itstranscriptional regulation is astonishingly limited. In this report,we used bioinformatics and loss-of-function approaches to show forthe first time that Sp1 regulates c-Abl transcription, expression andthe proliferation in smooth muscle cells. Since c-Abl is ubiquitouslyexpressed in human cells, Sp1-mediated c-Abl transcription couldbe common.

We used site-directed mutagenesis to disrupt the five GC-richdomains and assessed the luciferase activity of individual mutants.We found that Site 1, 4 or 5 affected the activity of c-Abl promoter.In contrast, Site 2 or 3 did not affect promoter activity significantly.

Fig. 5. ERK1/2 regulates Sp1, c-Abl and cellproliferation. (A) HASM cells were treated with10 ng/ml PDGF for 10 min in the absence orpresence of 10 µM the MEK inhibitor U0126 orleft unstimulated. The level of ERK1/2phosphorylation (ERK1: Thr-202/Tyr 204; ERK2:Thr-185/Tyr-187, p-ERK1/2) was determinedby immunoblot analysis. Data are mean±s.d.(n=4–7). *P

These results imply that not all binding sites are critical for promoteractivation. This could be due to inaccessibility of certain bindingsites on promoters in vivo (Soufi et al., 2015). In addition, thelocation of DNA-binding motifs could also influence the interactionof transcription factors with promoters (Westholm et al., 2008).We also discovered that growth factor activation coordinately

promotes the occupancy of Sp1 on the endogenous c-Abl promoterand c-Abl expression in smooth muscle cells. As such, growth factor-mediated c-Abl upregulation ismodulated through c-Abl transcriptionin cells. However, expression of miR-203 (a microRNA thatdownregulates c-Abl) is reduced in T-cell tumors (Bueno et al.,2008) and asthmatic human airway smoothmuscle cells (Cleary et al.,2013; Liao et al., 2015). Moreover, growth factor concentration orsignaling is upregulated in asthma (Ammit and Panettieri, 2003; Booyet al., 2011).We do not rule out the possibility thatmiR-203 reductionmay be also involved in growth factor-induced c-Abl expression.Sp1 has been implicated in the regulation of the vascular

endothelial growth factor promoter, and phosphorylation of Sp1 atThr-453 and Thr-739 by ERK1/2 increases Sp1 promoter activity inSL2 Drosophila cells (Milanini-Mongiat et al., 2002). BecausePDGF stimulation increases ERK1/2 phosphorylation (Jia et al.,2012; Wang et al., 2013a) (Fig. 1B), we evaluated the effects ofPDGF stimulation on Sp1 phosphorylation. We unexpectedly foundthat PDGF activation did not induce Sp1 phosphorylation at thesetwo residues. Thus, it is less likely that Thr-453 and Thr-739 have arole in modulating Sp1 activity in this cell type. This discrepancymay stem from different cell types and/or different experimentalconditions (growth factors versus inducible Raf-1 activation).Intriguingly, we found that Sp1 total protein was upregulated by

PDGF in smooth muscle cells. The time course results suggest thatERK1/2 activation precedes the upregulation of Sp1 and c-Abl.Furthermore, ERK1/2 inhibition attenuated Sp1 expression, c-Ablexpression and cell proliferation. These results led us to suggest thatERK1/2 activation may upregulate Sp1 expression, which maysubsequently promote c-Abl expression and cell proliferation.

The mechanisms by which ERK1/2 modulates Sp1 expressionare currently unknown. ERK1/2 has been shown to phosphorylateand regulate E20 transformation-specific family transcriptionfactors (Roberts and Der, 2007). Thus, ERK1/2 could regulateunidentified transcription factors, which in turn control Sp1expression. Activated ERK1/2 has also been implicated in non-coding RNA regulation (e.g. miRNA) (Qu et al., 2017). ERK1/2could control unidentified non-coding RNAs that modify Sp1expression. Future studies are required to test these possibilities.

Our previous studies have demonstrated that c-Abl promotesERK1/2 phosphorylation by activating the Raf1–MEK1/2 pathway(Jia et al., 2012;Wang et al., 2013a). In this study, we verified that c-Abl positively regulates ERK1/2 activation. Moreover, c-Ablregulated Sp1 expression in cells. Thus, we propose that c-Abltyrosine kinase conversely regulates ERK1/2 activation and Sp1expression in cells during growth factor stimulation.

In conclusion, we provide the first evidence that Sp1 regulates c-Abl tyrosine kinase at the transcription level. We also unveiled anovel transcriptional regulation of c-Abl. In response to growthfactor activation, Sp1 increases c-Abl promoter activity, whichsubsequently enhances c-Abl expression. Conversely, c-Ablregulates ERK1/2 activation and Sp1 expression (Fig. 7). Hence,Sp1, c-Abl and ERK1/2 form a regulatory loop.

Fig. 6. c-Abl conversely regulatesSp1 expression. (A) HASM cellsexpressing control shRNA or stablec-Abl KD cells were treated with10 ng/ml PDGF for 24 h, or leftuntreated. Protein expression wasdetermined by immunoblot analysis.c-Abl expression after varioustreatments is normalized to that in cellsnot treated with PDGF. c-Abl KD isverified in these cells. Sp1 expressionafter various treatments is normalizedto that in cells without treatment ofPDGF. The data represent means±s.d. (n=5–6). *P

MATERIALS AND METHODSCell cultureHuman airway smooth muscle (HASM) cells were prepared from humanbronchi and adjacent tracheas obtained from the International Institute forAdvanced Medicine (Wang et al., 2014a,b, 2013a,b, 2015). Human lungswere non-transplantable, and informed consented was obtained from allsubjects for research. This study was approved by the Albany MedicalCollege Committee on Research Involving Human Subjects. All clinicalinvestigation have been conducted according to the principles expressed inthe Declaration of Helsinki. Briefly, muscle tissues were incubated for20 min with dissociation solution [130 mM NaCl, 5 mM KCl, 1.0 mMCaCl2, 1.0 mMMgCl2, 10 mMHepes, 0.25 mMEDTA, 10 mMD-glucose,10 mM taurine, pH 7, 4.5 mg collagenase (type I), 10 mg papain (type IV),1 mg/ml BSA and 1 mM dithiothreitol]. All enzymes were purchased fromSigma-Aldrich. The tissues were then washed with Hepes-buffered salinesolution (composition in mM: 10 Hepes, 130 NaCl, 5 KCl, 10 glucose, 1CaCl2, 1 MgCl2, 0.25 EDTA, 10 taurine, pH 7). The cell suspension wasmixed with Ham’s F12 medium supplemented with 10% (v/v) fetal bovineserum (FBS) and antibiotics (100 units/ml penicillin, 100 µg/mlstreptomycin). Cells were cultured at 37°C in the presence of 5% CO2 inthe same medium. The medium was changed every 3–4 days until cellsreached confluence, and confluent cells were passaged with trypsin/EDTAsolution (Li et al., 2006; Wang et al., 2013a). Smooth muscle cells from fournon-asthmatic subjects were used for the experiments. In some cases,duplicated experiments were performed for cells from a donor. Cells wererecently authenticated by morphological analysis and immunoblotting/immunostaining of α-actin. No contamination was found for these cells.

Immunoblot analysisCells were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2%SDS, 80 mM Tris-HCl pH 6.8, 10% glycerol and 0.01% BromophenolBlue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes. Themembranes were blocked with bovine serum albumin or milk for 1 h andprobed with use of primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (ThermoFisher Scientific). Proteins werevisualized by enhanced chemiluminescence (ThermoFisher Scientific)using the GE Amersham Imager 600 system.

Anti-total Sp1 antibody (1:1000) was purchased from Invitrogen(#PA529165, L/N RJ2284607). Anti-c-Abl antibody (1: 1000) was

purchased from Cell Signaling (#2862S, L/N 15). Anti-Sp1 and anti-c-Ablantibodies were validated by using corresponding knockdown cells. Anti-phospho-Sp1 (Thr-453) antibody (1:500) was purchased from ThermoFisherScientific (#PA5-19658, L/N RH-2250163) and validated by examining themolecular mass of detected bands. Anti-phospho-Sp1 (Thr-739) antibody(1:500) was purchased from Life Span Biosciences Inc. (#LS-C205086, L/N59177) and validated by examining molecular mass of detected bands.Anti-total vimentin antibody (1:1000) was custom-made as previouslydescribed (Li et al., 2006; Tang et al., 2005) and validated by examining themolecular mass of detected bands. Anti-α-tubulin antibody (1:1000) waspurchased from Santa Cruz Biotechnology (#SC-32293, L/N G0114).Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody(1:1000) was acquired from Santa Cruz Biotechnology (#SC-32233,K0315). Anti-phospho-ERK1/2 antibody (1:1000) was purchased from CellSignaling (#4370S, L/N 38). Anti-total ERK1/2 antibody (1:1000) waspurchased fromCell Signaling (#4695S, L/N 14). Antibodies againstα-tubulin,GAPDH, phospho-ERK1/2 and total ERK1/2 were validated by the examiningmolecular mass of detected bands. Finally, vendors have provided data sheetsto show that antibodies were validated by positive controls.

The levels of proteins were quantified by scanning densitometry ofimmunoblots (Fuji Multigauge Software or GE IQTL software). Theluminescent signals from all immunoblots were within the linear range.

Cell knockdown and cell transfectionFor Sp1 knockdown, control siRNA (SC-37007/Lot# B1517) and Sp1siRNA (SC-29487/Lot# J0616) were purchased from Santa CruzBiotechnology. HASM cells were transfected with siRNA according tothe manual of the manufacturer (Santa Cruz Biotechnology). Stable c-Ablknockdown and corresponding control cells were generated using lentivirusencoding control or c-Abl shRNA and cultured as previously described(Wang et al., 2014a, 2013a, 2018). For protein expression, cells weretransfected with pN3-Sp1FL (Addgene #24543) or pEGFP (control,Clontech) using the FuGene HD transfection reagent (Promega).

Assessment of c-Abl promoter activityc-Abl promoter reporter (p-EZX-PG04) was purchased from GeneCopoeia(MD). The reporter uses a dual-reporter system, which uses GaussiaLuciferase (GLuc) as the promoter reporter and SeAP (secreted alkalinephosphatase) as the internal control for signal normalization. Cells weretransfected with the reporter plus siRNAs or expression constructs using theFuGene HD transfection reagent. They were then cultured in the growthmedium for 48–72 h. The luciferase activity was determined by using thesecrete-pair dual luminescence assay kit (Gene Copoeia, #LF032).

Assessment of DNA synthesis and cell proliferationCells were treated with human platelet-derived growth factor (PDGF)-BB(Sigma, 10 ng/ml) in F12 medium containing 0.25% FBS. Additional cellswere cultured in the mediumwith 0.25% FBS as a control. The 5′-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit (Millipore) was used toevaluate DNA synthesis. BrdU is an analog of thymidine, which is able to beincorporated into newly synthesized DNA. Cells in 96-well plates weretreated with BrdU for 24 h. They were fixed and reacted with BrdU antibodyfollowed by incubation with secondary antibody conjugated to peroxidase.They were then reacted with peroxidase substrates. The reaction wasdetected using a Promega GloMax-Multi Microplate reader. The number ofviable cells were evaluated with a Trypan Blue exclusion test. Triplicatedsamples were averaged for each experiment (Jiang and Tang, 2015).

Assessment of mRNA expressionTotal RNA from smooth muscle cells was purified by using the GeneJETRNA Purification Kit (Thermo Scientific) according to the manufacturer’sinstructions. Reverse transcription was performed using the iScript cDNASynthesis Kit (Bio-Rad). Quantitative real-time PCR was performed byusing the SsoAdvancedUniversal SYBRGreen Supermix kit (Bio-Rad) anda real-time PCR system (Bio-Rad). For the detection of human c-Abl mRNA,the 5′-primer sequence was 5′-AGCTCTACGTCTCCTCCGAG-3′; the 3′-primer sequence was 5′-CAGCTTGTGCTTCATGGTGA-3′. Human β2-microglobulin (B2M) mRNAwas used as a control. The 5′-primer sequence

Fig. 7. Activation loop between ERK1/2, Sp1 and c-Abl. In response toactivation with growth factors, Sp1 increases c-Abl promoter activity,which enhances c-Abl expression. Conversely, c-Abl regulates ERK1/2activation and Sp1 expression.

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RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs222380. doi:10.1242/jcs.222380

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https://www.addgene.org/24543/

of B2M was 5′-TGCTGTCTCCATGTTTGATGTATCT-3′; the 3′-primersequence of B2M was 5′-TCTCTGCTCCCCACCTCTAAGT-3′. Theexpression level of c-Abl mRNA was expressed as the ratio of c-AblmRNA to that of B2M mRNA.

Site-directed mutagenesisDifferent c-Abl promoter reporter mutants were generated by usingQuikChange II XL site-directed mutagenesis kit (Agilent Technologies) aspreviously described (Jiang and Tang, 2015). The construct (p-EZX-PG04)was used as a template. For mutant 1, the sequence of forward primer was 5′-CCAGAGCCGGGAGGTTCGGCGGTGTCCCGGG-3′. The sequence ofreverse primer was 5′-CCCGGGACACCGCCGAACCTCCCGGCTCTGG-3′. For mutant 2, the sequence of forward primer was 5′-CCGGACG-TCGCCGTGTTCGGGGCCGAGGGCGG-3′. The sequence of reverseprimer was 5′-CCGCCCTCGGCCCCGAACACGGCGACGTCCGG-3′.For mutant 3, the sequence of forward primer was 5′-GTGGGCGGGG-CCGAGTTCGGGGCCTGGCCTCG-3′. The sequence of reverse primerwas 5′-CGAGGCCAGGCCCCGAACTCGGCCCCGCCCACG-3′. Formutant 4, the sequence of forward primer was 5′-CCCCTACCGGCGG-GGTTCGGCTGGGTCCCTCGG-3′. The sequence of reverse primer was 5′-CCGAGGGACCCAGCCGAACCCCGCCGGTAGGGG-3′. For mutant 5,the sequence of forward primer was 5′-GATGTGACTGCCTGAGTTC-GGTGGTGGTGTCAGC-3′. The sequence of reverse primer was 5′-GCTGACACCACCACCGAACTCAGGCAGTCACATC-3′. Plasmids werepurified by using the QIAPrep Spin Miniprep kit (Qiagen, Germany). DNAsequencing was performed by Genewiz.

ChIP analysisChIP assay was performed according to the protocol at Aparicio et al. (2004)with modifications. Briefly, adherent cells were incubated in culturemedium containing 1% formaldehyde with gentle shaking for 10 min atroom temperature and crosslinking was stopped by addition of 2.5 Mglycine to a final concentration of 0.125 M glycine. After two washes withcold PBS, cells were harvested in ice cold lysis buffer (10 mM Tris-HCl pH8.0, 85 mM KCl, 0.5% NP-40, 5 mM EDTA and fresh proteinase inhibitorcocktail) and incubated on ice for 10 min. Nuclei were collected andsuspended in cold RIPA buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl,0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 5 mMEDTA andfresh proteinase inhibitor cocktail). The mixtures were sonicated to shear thegenomic DNA to an average of 200–300 bp. Cleared extracts were blockedwith protein A/G beads (Upstate Biotechnology) and aliquots of thesupernatants were used for immunoprecipitation with the anti-Sp1 antibodyor IgG. After seven washes by RIPA buffer with gentle rotation for 5 mineach time, the proteins were eluted from the beads by 0.5 ml elution buffer(0.1 M NaHCO3 and 1% SDS). The DNA samples were recovered byphenol extraction and ethanol precipitation after reversal of crosslinking.The purified DNA was then analyzed by PCR within linear amplificationrange followed by agarose gel electrophoresis. The sequence of forwardprimer was 5′-GGGAAAGCGGCTCTTGGG-3′. The sequence of reverseprimer was 5′-TCAGGCACAGACACCAAAC-3′.

Statistical analysisAll statistical analysis was performed using Prism software (GraphPadSoftware, San Diego, CA). Differences between pairs of groups wereanalyzed by Student–Newman–Keuls test or the Dunn’s method. Comparisonamong multiple groups was performed by one-way or two-way ANOVAfollowed by a post hoc test (Tukey’s multiple comparisons). Values of n referto the number of experiments used to obtain each value. Power and SampleSize Calculation software (Vanderbilt University, http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize) was used to determine the sample size.P

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RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs222380. doi:10.1242/jcs.222380

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