Transforming Growth Factor-beta 1 Stimulates Protein Kinase A in Mesangial Cells*

Lewei Wang, Yanqing Zhu, and Kumar SharmaDagger

From the Department of Medicine, Division of Nephrology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We recently demonstrated that transforming growth factor-beta (TGF-beta ) stimulates phosphorylation of the type I inositol 1,4,5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta 1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 µM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta 1 treatment. TGF-beta 1 was also found to stimulate translocation of the alpha -catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta 1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 µM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta 1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Transforming growth factor-beta (TGF-beta )1 has been established as an important cytokine intimately involved in tissue fibrosis. In a variety of kidney diseases characterized by excess glomerular matrix deposition, TGF-beta has been found to be up-regulated (1). Diabetic kidney disease is characterized by accumulation of a variety of matrix molecules, including fibronectin (2). We have demonstrated that TGF-beta 1 is stimulated in the diabetic kidney and that neutralization with anti-TGF-beta antibodies significantly inhibits diabetes-induced fibronectin gene expression (3). Although stimulation of fibronectin by TGF-beta is well recognized (4, 5), the signaling pathways by which TGF-beta regulates fibronectin gene expression are poorly understood.

Recent studies have demonstrated that TGF-beta initially binds to its type II receptor and then forms a heteromeric complex with the type I receptor (reviewed in Ref. 6). Cross-phosphorylation of the type I receptor by the type II receptor is critical for subsequent phosphorylation of various members of the recently described Smad family of proteins. Phosphorylation of Smad2 and Smad3 has been demonstrated to play an important role in mediating the effects of TGF-beta on cell proliferation (7, 8); however, their role in stimulation of matrix molecules such as fibronectin is unclear. The protein kinase A (PKA) pathway induces many effects on cells similar to TGF-beta , and in particular, activation of both TGF-beta and PKA stimulates fibronectin production, at least in part, by stimulating gene transcription (4). A critical step involved in the transcriptional gene regulation by the PKA pathway is the phosphorylation of the cAMP response element-binding protein (CREB). Interestingly, CREB phosphorylation by TGF-beta has been demonstrated in several cell types (9-11), and the consensus cAMP response element (CRE, TGAGTCA) has been implicated in mediating the effects of TGF-beta on the fibronectin promoter in rat mesangial cells (12) and on the cyclin A promoter in Chinese hamster lung fibroblasts (13). Although these findings would suggest that the PKA pathway is involved in TGF-beta signaling, this possibility was considered unlikely primarily because cAMP levels were not elevated following TGF-beta treatment (9, 10).

PKA is composed of two regulatory subunits and two catalytic subunits in its inactive state (14). Elevation of cAMP levels leads to dissociation of the heteromeric complex, thus allowing the free catalytic subunit to be active as a serine/threonine kinase in the cytoplasm and nucleus. Until recently, activation of PKA without a rise in cAMP levels was not thought to occur. In an elegant study by Zhong et al. (15), the alpha -catalytic subunit of PKA was found to be bound to Ikappa B in the cytoplasm, rather than by one of its regulatory subunits; degradation of Ikappa B led to activation of the alpha -catalytic subunit. Therefore, it is apparent that other mechanisms are operant to allow for PKA activation independently of cAMP. In support of a role for the PKA pathway being involved in TGF-beta signaling, we recently demonstrated that TGF-beta -induced phosphorylation of the type I inositol 1,4,5-trisphosphate receptor in mesangial cells appeared to be mediated by PKA (16). In the present study, we demonstrate that TGF-beta stimulates PKA activity without elevating intracellular cAMP levels in mesangial cells and that inhibition of PKA attenuates TGF-beta -induced stimulation of CREB phosphorylation and fibronectin gene expression.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [gamma -32P]ATP was purchased from NEN Life Science Products. An enhanced chemiluminescence system was purchased from Amersham Pharmacia Biotech. TGF-beta 1 was purchased from R&D Systems (Minneapolis, MN). All other reagents were from Sigma unless otherwise noted.

Cell Culture-- An SV40-transformed murine glomerular mesangial cell line, which has been previously described (17), was primarily used in these studies. These cells retain many of the differentiated characteristics of mesangial cells in primary culture. We also performed a limited series of experiments in rat glomerular mesangial cells that were conducted between passages 4 and 6 and in the mink lung epithelial cell line (CCL-64, obtained from American Type Culture Collection, Rockville, MD).

In Vitro Kinase Assay for PKA Activity-- Murine mesangial cells (MMCs) were grown to confluence in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum and then growth-arrested for 24 h in serum-free DMEM. Cells were treated with agonists for various periods of time, washed with PBS, and harvested with cold extraction buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM beta -mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotonin, 0.5 mM phenylmethylsulfonyl fluoride. Protein concentrations of the crude lysates were quantitated, and equal amounts of protein were added to a reaction mixture containing 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml bovine serum albumin, 100 µM biotinylated PKA peptide substrate (Kemptide, LRRASLG; Promega, Madison, WI), 3000 Ci/mmol [gamma -32P]ATP, and 0.5 mM ATP per reaction. Experiments were performed in parallel with the addition of a PKA inhibitor peptide (TTYADFIASGRTGRRNAIHD, 1 µM; Promega). The reaction was allowed to proceed for 5 min at 30 °C and then terminated by the addition of 2.5 M guanidine hydrochloride. Five µl of sample were spotted onto streptavidin-coated discs, washed repeatedly, dried in an oven, and placed in scintillation vials for radioactive counting. To visually demonstrate that the Kemptide peptide was indeed phosphorylated, the in vitro kinase reaction was also performed with fluorescently tagged Kemptide (Promega). Phosphorylation of this peptide alters the peptide's net charge from +1 to -1, allowing the phosphorylated peptide to be separated from the unphosphorylated peptide on an agarose gel at neutral pH.

Transfection of MMCs with Protein Kinase A Inhibitor (PKI) Expression Vector-- The expression vectors for PKI and mutant PKI driven by the Rous sarcoma virus promoter were obtained from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR) (18). The mutant PKI sequence codes for glycine rather than arginine at positions 20 and 21. The arginines at these sites are essential for inhibition of catalytic subunit activity. Subconfluent MMCs were transfected with 10 µg of plasmid and Superfect transfection reagent (QIAGEN Inc., Chatsworth, CA) for 3 h. Cells were washed and rested in serum-free DMEM for an additional 16 h prior to the addition of agonists.

Western Blot Analysis-- MMCs were treated with TGF-beta and/or the PKA inhibitor H-89 (3 µM, 30 min) prior to the addition of TGF-beta in serum-free DMEM. Cell extracts were prepared as described previously (16). Nuclear extracts were prepared according to the method of Schreiber et al. (19). Total cellular or nuclear protein was quantitated using the Bio-Rad DC protein assay, and 20 µg of protein were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Immunoblotting was performed with antibodies against the alpha -catalytic subunit of PKA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-phospho-CREB antibody (Upstate Biotechnology, Inc., Lake Placid, NY), anti-CREB antibody, anti-Ikappa B-alpha antibody, or anti-NF-kappa B p65 antibody (Santa Cruz Biotechnology, Inc.). Immunoreactive bands were detected using the enhanced chemiluminescence system.

Confocal Analysis-- MMCs were grown in 6-well tissue culture dishes on No. 1 coverslips pretreated with poly-D-lysine (0.1 mg/ml for 5 min) with DMEM containing 10% fetal calf serum. After cells were adherent, they were rested in serum-free DMEM overnight and then exposed to TGF-beta 1 (10 ng/ml) or forskolin (10 µM) for 15 min. After exposure to agonists, cells were washed with PBS three times and fixed with 3.7% formaldehyde for 10 min at room temperature. After repeated washing, cells were permeabilized with 0.05% Triton X-100 in PBS (PBS/TX) for 10 min at room temperature, washed with PBS/TX three times, blocked with 4% normal goat serum in PBS/TX for 10 min, and then incubated with anti-alpha -catalytic antibody (1:200) for 30 min at 37 °C. Cells were washed with PBS/TX and blocked again with 4% normal goat serum, and secondary antibody (fluorescein-conjugated goat anti-rabbit antibody, 1:500 dilution; Rockland Inc., Gilbertsville, PA) was applied for 30 min at 37 °C. After repeated washing, cells were post-fixed with 3.7% formaldehyde for 10 min at room temperature and washed, and coverslips were placed on glass slides and mounted with SlowFade (Molecular Probes, Inc., Eugene, OR). Slides were visualized with a confocal microscope (courtesy of Dr. James Keen, Thomas Jefferson University), and representative regions were photographed. Control cells, stained only with secondary antibody, showed minimal fluorescence.

Measurement of cAMP-- Cyclic AMP levels were measured in MMCs after treatment with TGF-beta 1 (10 ng/ml; 5, 15, and 30 min) or forskolin (10 µM, 15 min) using a Biotrak cAMP enzyme immunoassay kit (Amersham Pharmacia Biotech). MMCs were trypsinized and resuspended in PBS with 65% (v/v) ethanol. The cell precipitates were centrifuged, the supernatants were drawn off, and the extracts were dried in a vacuum oven. Extracts were resuspended in assay buffer, acetylated, and assayed for cAMP following the instructions supplied by the manufacturer. MMCs were assayed in the absence and presence of isobutylmethylxanthine (100 µM) 10 min prior to the addition of TGF-beta 1 or forskolin.

Northern Analysis-- To assess whether TGF-beta 1-induced fibronectin mRNA expression was affected by inhibition of PKA, MMCs transiently transfected with PKI expression vector or mutant PKI were treated with TGF-beta 1 (10 ng/ml) for 24 h and washed with ice-cold PBS, and total RNA was isolated using acid guanidinium thiocyanate/phenol/chloroform (20). Twenty µg of total RNA were loaded onto a 1.2% agarose gel containing 2.2 M formaldehyde, electrophoresed, and transferred onto nylon membrane. The probe for murine fibronectin has been described previously (3). Hybridization and washing conditions were performed as described previously (21). To standardize for loading, membranes were stripped and reprobed with a beta -actin cDNA probe (kindly provided by Dr. Pamela A. Norton). Densitometric analysis was performed as described previously (16), and mRNA levels were calculated relative to those of beta -actin.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

TGF-beta Stimulates PKA Activity-- MMCs were treated with TGF-beta 1 for various time points, and an in vitro kinase assay was performed utilizing a biotinylated substrate for PKA (Fig. 1A). The reaction mixture was spotted onto avidin-coated discs to bind only the biotinylated peptide, thus increasing the specificity of the assay. With 15 min of administration of TGF-beta 1, PKA activity was increased by 3-fold relative to control values and remained elevated at 30 min. The addition of a specific peptide inhibitor of the catalytic subunit of PKA (PKI) completely blocked the increased kinase activity in TGF-beta -treated samples, demonstrating that the increased kinase activity was specific for PKA (Fig. 1A). A dose-response relationship was noted, with maximal stimulation of PKA noted at a concentration of 10 ng/ml TGF-beta 1 (Fig. 1B). To further demonstrate that PKA activity was being stimulated by TGF-beta 1 or forskolin, cells were transiently transfected with an expression vector for the PKI peptide to inhibit PKA activity or a PKI peptide that was mutated in its PKA catalytic recognition site. TGF-beta or forskolin stimulated PKA activity in cells expressing the mutant PKI peptide, but not in MMCs expressing wild-type PKI (Fig. 1C). To visually demonstrate that the peptide substrate for PKA was indeed phosphorylated by TGF-beta 1, a fluorescently tagged peptide substrate was employed in the in vitro kinase assay. Phosphorylation of the peptide promotes migration to the positive electrode on an agarose gel. TGF-beta 1 treatment for 5 and 15 min (lanes 3 and 4) stimulated migration of this peptide to the positive electrode (Fig. 1D).


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Fig. 1.   TGF-beta induces PKA activation. A, time course. MMCs were treated with TGF-beta 1 (10 ng/ml) for the indicated time periods, and PKA activity was measured as described under "Experimental Procedures." Cell lysates were added to a reaction mixture containing [gamma -32P]ATP and a biotinylated peptide specific for PKA. As an additional control for specificity, the PKI peptide (1 µM) was added in parallel samples. Results are expressed as % of control values with means ± S.E. from three separate experiments. B, dose response. MMCs were treated with the indicated doses of TGF-beta 1 for 30 min, and PKA activity was assessed. PKA activity is expressed as pmol/min/mg of [32P]phosphate transferred to Kemptide substrate that was inhibited by PKI (1 µM). Experiments were repeated twice with essentially the same results. C, MMCs transfected with an expression vector for mutant PKI (left) or PKI (right) were treated with TGF-beta 1 (10 ng/ml, 30 min), and PKA activity was assessed. Results are expressed as means ± S.E. from a representative experiment. Experiments were repeated twice with essentially the same results. *, p < 0.05 compared with values for the corresponding controls in A-C. D, visual demonstration of Kemptide phosphorylation by TGF-beta 1 treatment. Shown are control samples (lanes 1 and 2), TGF-beta 1 treatment (10 ng/ml) for 15 min (lane 3), TGF-beta 1 treatment for 30 min (lane 4), and forskolin treatment (10 µM) for 15 min (lane 5) using a fluorescently tagged Kemptide substrate as described under "Experimental Procedures." Unphosphorylated Kemptide bands are noted above the origin, and phosphorylated Kemptide bands are noted below the origin. Experiments were repeated twice with essentially the same results.

To determine whether TGF-beta 1 stimulates kinase activity in other cell types, we evaluated nontransformed rat mesangial cells (between passages 4 and 6) and the mink lung epithelial cell line, which has commonly been found to be very sensitive to TGF-beta . PKA activity was increased by ~1.5-fold in rat mesangial cells and by ~2-fold in mink lung epithelial cells treated with TGF-beta 1 (Fig. 2, A and B).


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Fig. 2.   TGF-beta stimulates PKA activity in rat mesangial cells and mink lung epithelial cells. Rat mesangial cells (A) and mink lung epithelial cells (B) were stimulated with TGF-beta 1 (10 ng/ml, 15 min), and PKA activity was assessed as described for Fig. 1B. Results are expressed as means ± S.E. from three separate experiments. *, p < 0.05 compared with values for the corresponding control.

TGF-beta Stimulates Translocation of alpha -Catalytic Subunit of PKA-- Activation of PKA leads to dissociation of the catalytic subunit from its regulatory subunit and translocation to the nucleus (14). To demonstrate that PKA is activated and that the catalytic subunit is dissociated from its inhibitory regulatory subunit, nuclear proteins were isolated from control and TGF-beta -treated MMCs, and immunoblot analysis was performed with an antibody against the alpha -catalytic subunit of PKA. Fig. 3 demonstrates accumulation of the catalytic subunit in the nucleus with TGF-beta 1 treatment. Confocal microscopy (Fig. 4) also demonstrated a redistribution of the alpha -catalytic subunit from a primarily perinuclear distribution in untreated MMCs (Fig. 4A) to a nuclear localization in TGF-beta 1-treated (Fig. 4B) and forskolin-treated (Fig. 4C) MMCs. As compared with control cells, TGF-beta 1- and forskolin-treated cells demonstrated a similar redistribution of the alpha -catalytic subunit diffusely into the cytoplasm.


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Fig. 3.   Translocation of the alpha -catalytic subunit of PKA to the nucleus with TGF-beta 1 stimulation of MMCs. Twenty µg of nuclear protein were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with the antibody for the alpha -catalytic subunit of PKA. Shown are representative data from a single experiment with two separate control samples (lanes 1 and 2), TGF-beta 1 (10 ng/ml) treatment for 15 min (lanes 3 and 4), and TGF-beta 1 treatment for 30 min (lanes 5 and 6). Experiments were repeated twice with essentially the same results.


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Fig. 4.   Translocation of the alpha -catalytic subunit of PKA by confocal microscopy. MMCs treated with TGF-beta 1 (10 ng/ml) or forskolin (10 µM) for 15 min were fixed on coverslips, and immunofluorescence was performed with the antibody for the alpha -catalytic subunit of PKA. Slides were observed by confocal microscopy, and representative areas were photographed. The location of the alpha -catalytic subunit in the basal state is in the perinuclear location as shown in A (control). With TGF-beta exposure, enhanced nuclear staining and diffuse cytoplasmic staining were observed (B). A very similar appearance occurred with exposure of MMCs to forskolin (C).

TGF-beta Does Not Increase Intracellular cAMP Levels or Affect NF-kappa B/Ikappa B-- Increased intracellular cAMP levels enhance binding of cAMP to the regulatory subunits of PKA and promote dissociation from the catalytic subunit. Therefore, cAMP levels were measured following TGF-beta 1 treatment after various time intervals. MMCs stimulated with TGF-beta 1 for 5, 15, or 30 min did not raise intracellular cAMP levels (Fig. 5). Forskolin treatment did cause a marked increase in cAMP levels in MMCs, as would be expected. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, TGF-beta also did not increase cAMP levels (data not shown).


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Fig. 5.   Cyclic AMP levels are not increased with TGF-beta treatment. Cyclic AMP levels were measured in MMCs after treatment with TGF-beta 1 (10 ng/ml; 5, 15, and 30 min) or forskolin (10 µM, 15 min) using a Biotrak enzyme immunoassay assay kit. Results are expressed as means ± S.E. from three separate experiments.

To evaluate if TGF-beta -induced activation of PKA may involve degradation of Ikappa B as a cAMP-independent mechanism of PKA activation (15), we analyzed protein levels of Ikappa B after TGF-beta treatment. In whole cell lysates, treatment of MMCs with TGF-beta 1 for 30 min did not reduce Ikappa B (Fig. 6A). Immunoblot analysis of nuclear protein from MMCs treated with TGF-beta 1 for 30 min did not reveal accumulation of NF-kappa B (Fig. 6B). Analysis by electrophoretic mobility shift assay also did not demonstrate enhanced binding of nuclear protein to a consensus NF-kappa B probe with TGF-beta 1 treatment for 30 min (data not shown). Therefore, it is unlikely that Ikappa B degradation is responsible for TGF-beta -induced stimulation of PKA alpha -catalytic subunit activation.


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Fig. 6.   Immunoblot analysis of Ikappa B and NF-kappa B in MMCs treated with TGF-beta . A, immunoblot analysis with anti-Ikappa B antibody of the whole cell lysate (20 µg of protein) derived from control MMCs and MMCs treated with TGF-beta 1 (10 ng/ml) for 30 min; B, immunoblot analysis with anti-NF-kappa B antibody of nuclear protein (20 µg) derived from control MMCs and MMCs treated with TGF-beta 1 (10 ng/ml) for 30 min.

TGF-beta -induced CREB Phosphorylation Is Blocked by Inhibition of PKA-- To determine the functional significance of TGF-beta -induced PKA activation, we evaluated if CREB was phosphorylated in response to TGF-beta 1 in MMCs by performing immunoblot analysis of nuclear protein with anti-phospho-CREB antibody. Fig. 7 demonstrates enhanced CREB phosphorylation (43-kDa band) in nuclear protein isolated from TGF-beta 1-treated cells as compared with control cells. Pretreatment of MMCs with H-89, a relatively specific inhibitor of PKA, was sufficient to completely inhibit TGF-beta -induced CREB phosphorylation. Immunoblot analysis of the same membrane with an antibody that recognizes total CREB revealed that TGF-beta 1 did not lead to accumulation of CREB in the nucleus (data not shown). Anti-phospho-CREB antibody is raised against the phosphoserine in a portion of the CREB protein that shares 100% homology with the phosphoserine site of the transcription factor ATF-1 (22); therefore, this protein may also be demonstrated on immunoblotting. The band at 38 kDa corresponds to phospho-ATF-1 and was also found to be markedly stimulated in nuclear extracts from TGF-beta 1-treated cells (Fig. 7). Inhibition of PKA by H-89 also completely prevented TGF-beta -induced phosphorylation of ATF-1. Treatment of MMCs transiently transfected with the PKI expression vector failed to demonstrate increased CREB or ATF-1 phosphorylation in response to TGF-beta 1 treatment (data not shown). Thus, CREB phosphorylation by TGF-beta 1 appears to be largely dependent on activation of PKA.


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Fig. 7.   Inhibition of PKA blocks TGF-beta -induced CREB phosphorylation. Shown are the results from the immunoblot analysis with anti-phospho-CREB antibody of nuclear extract from MMCs treated with TGF-beta 1 (10 ng/ml, 30 min) alone or in the presence of the PKA inhibitor H-89 (3 µM, 30 min prior to TGF-beta 1 treatment). The upper bands correspond to phospho-CREB (P-CREB), and the lower bands correspond to phospho-ATF-1 (P-ATF). Data are representative of a single experiment. Experiments were repeated twice with essentially the same results.

TGF-beta -induced Fibronectin mRNA Stimulation Is Attenuated by Inhibition of PKA-- Fibronectin gene stimulation is considered to be controlled, at least in part, by CREs located in the fibronectin promoter, and both TGF-beta and PKA stimulation may stimulate the fibronectin promoter via CRE sites (4, 12, 23). To assess if PKA may be involved in stimulation of steady-state fibronectin mRNA levels by TGF-beta , we performed Northern analysis of MMCs transiently transfected with an expression vector for either wild-type PKI or mutant PKI (Fig. 8). MMCs overexpressing mutant PKI treated with TGF-beta 1 for 48 h demonstrated a 2.5-fold stimulation of steady-state fibronectin mRNA levels (lane 4 as compared with lane 3), whereas MMCs overexpressing wild-type PKI had a blunted effect (lane 2 as compared with lane 1) with TGF-beta 1 treatment (Fig. 8).


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Fig. 8.   MMCs transfected with PKI expression vector inhibit TGF-beta -induced fibronectin gene expression. Shown are the results from a representative Northern analysis of total RNA (20 µg) from MMCs transfected with an expression vector for PKI (lanes 1 and 2) or with a mutant PKI expression vector (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of TGF-beta 1 (10 ng/ml, 24 h) hybridized with a cDNA probe for fibronectin (FN). The blot was stripped and reprobed with beta -actin cDNA to standardize the amount of RNA loaded. Experiments were repeated twice with essentially the same results.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Our major conclusions are that TGF-beta 1 stimulates PKA in mesangial cells and that the PKA pathway plays an important role in TGF-beta 1-induced CREB phosphorylation and TGF-beta stimulation of fibronectin gene expression. Surprisingly, we found PKA activation by TGF-beta in the absence of a detectable rise in cAMP levels.

Our results demonstrating TGF-beta -induced activation of PKA are in contrast to other published reports in different cell types. Prior studies excluded the PKA pathway in TGF-beta 1 signaling in mink lung epithelial cells and rat mesangial cells primarily because TGF-beta did not elevate intracellular cAMP levels (9, 10). Separate studies in Chinese hamster ovary cells (13) and murine embryonic palatal cells (11), using overexpression of the regulatory subunit to block PKA activity (13) and a competitive analogue of cAMP ((Rp)-cAMP) (11), respectively, concluded that TGF-beta -induced CREB phosphorylation occurred independently of the PKA pathway. However, the recent demonstration by Zhong et al. (15) that PKA may be activated without cAMP elevation sets an important precedent that may provide an explanation to justify the different results. This group reported that the alpha -catalytic subunit of PKA is constitutively associated with Ikappa B, likely via an ankyrin repeat region of Ikappa B, and degradation of Ikappa B leads to stimulation of alpha -catalytic subunit kinase activity. These authors further suggested that other proteins containing ankyrin repeat sites may also function to bind the alpha -catalytic subunit of PKA and to regulate its activity independently of intracellular cAMP. Therefore, failure to observe an increase in cAMP-mediated kinase activity is not adequate evidence to exclude the involvement of the PKA pathway in a given cellular process.

It should be noted that in two of the above-mentioned studies (11, 13), PKA activity was measured by in vitro kinase assays after 10-15 min of exposure to TGF-beta and was found not to be increased. The in vitro kinase assays utilized either Kemptide substrate peptide (13) or a nonspecific peptide (11) as a phosphate acceptor from [32P]ATP with subsequent washing on phosphocellulose filters. The net phosphorylation detected would reflect the amount of phosphorylated target peptide substrate that binds to the phosphocellulose filter as well as other proteins that are present in the cell lysate that have basic residues (24). In addition, overall phosphatase activity in the cell lysate may also be reflected in this assay. As TGF-beta has been shown to activate serine/threonine kinases (6, 25) as well as various phosphatases (26), this assay method may not be conclusive. Our studies used two separate in vitro kinase assays wherein the Kemptide substrate is biotinylated or fluorescently labeled, thus assuring that phosphorylation of only the peptide substrate of interest would be measured. To enhance specificity, only the PKI-inhibitable fraction of kinase activity was taken to reflect PKA activity. In addition, we demonstrate translocation of the alpha -catalytic subunit to the nucleus with TGF-beta treatment by immunoblot analysis as well as confocal microscopy, providing an independent means of demonstrating PKA activation.

The mechanism(s) by which TGF-beta stimulates PKA activation remains unclear. Our studies failed to demonstrate that Ikappa B protein underwent degradation in whole cell lysate or that NF-kappa B was translocated to the nucleus with TGF-beta treatment. This would suggest that TGF-beta may affect degradation of another protein that associates with and inhibits the alpha -catalytic subunit of PKA, possibly via ankyrin repeat sites. In a study in which platelet-derived growth factor-BB was found to stimulate PKA without raising intracellular cAMP levels (27), it was suggested that phosphorylation of regulatory subunits may affect affinity of cAMP. Additionally, it is conceivable that localized intracellular compartments of cAMP may increase in response to TGF-beta , without a concomitant increase in total cellular cAMP. These possibilities are presently being explored in our laboratory.

TGF-beta -induced PKA activation likely contributes to CREB and ATF-1 phosphorylation. CREB phosphorylation by TGF-beta has been demonstrated by several independent studies (9-11). In a study employing murine embryonic palatal cells (11), TGF-beta -induced phosphorylation of CREB was demonstrated with an anti-phospho-CREB antibody specific for serine 133 (22). Serine 133 is not only the site of CREB phosphorylation by PKA, but also the site of CREB phosphorylation by mitogen-activated protein kinase-stimulated CREB kinase and calcium-calmodulin kinase. Evidence against the role of the latter two pathways in mediating TGF-beta -induced CREB phosphorylation was provided in the above study (11). The present study demonstrates that TGF-beta -induced CREB phosphorylation, employing the same anti-phospho-CREB antibody, is completely attenuated in mesangial cells when pretreated with the relatively specific inhibitor of PKA, H-89. Of note, H-89 inhibits the catalytic activity of PKA and is therefore not dependent on regulatory subunit binding of the catalytic subunit. Similar results were also found with the PKI peptide, which also binds to the free catalytic subunit of PKA. In addition, the phospho-CREB antibody also recognizes phospho-ATF-1 (22), and this nuclear transcription factor was also found to be phosphorylated by TGF-beta 1 treatment and reversed with PKA inhibition. The role of CREB phosphorylation in mediating gene regulation by TGF-beta is unclear; however, a recent study in Drosophila demonstrated that a CREB-binding site was important in mediating decapentaplegic (the Drosophila homologue of TGF-beta ) stimulation of the homeotic gene Ultrabiothorax (28).

TGF-beta 1 stimulation of the fibronectin promoter appears to require a consensus CRE (TGACGTCA) site (4, 23), and both CREB and ATF-1 derived from mesangial cells bind to this site (10). CREB phosphorylation enhances CREB binding to CREB-binding protein, thus activating gene transcription via the CRE (29). Inhibiting CREB phosphorylation would thus inhibit CREB binding to CREB-binding protein and may inhibit TGF-beta -induced stimulation of fibronectin gene transcription. Our finding that inhibition of PKA in mesangial cells (by overexpressing PKI) prior to TGF-beta treatment attenuates stimulation of fibronectin mRNA levels supports this hypothesis. The role of other transcription factors that are regulated by PKA and that bind to the CRE of the fibronectin promoter, such as ATF-1 and ATF-2, may also be relevant to TGF-beta stimulation of fibronectin gene transcription. In addition, it is likely that cross-talk among the PKA pathway, the mitogen-activated protein kinase pathway, and the Smad pathway participates in mediating specific responses to TGF-beta in various cell types.

    ACKNOWLEDGEMENT

We thank Gyorgy Hajnoczky for critical review of this manuscript.

    FOOTNOTES

* This work was supported by Grant KO8 DK02308 (to K. S.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Medicine, Div. of Nephrology, Thomas Jefferson University, Suite 353, JAH 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-6950; Fax: 215-923-7212; E-mail: sharma1{at}jeflin.tju.edu.

1 The abbreviations used are: TGF-beta , transforming growth factor-beta ; PKA, protein kinase A; CREB, cAMP response element-binding protein; CRE, cAMP response element; MMCs, murine mesangial cells; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PKI, protein kinase A inhibitor.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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