Mars entry guidance for mid-lift-to-drag ratio vehicle with control constraints

Kidney International, VoL 48 (1995), pp. 422—430

Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF- bioactivity in mesangial cells REBECCA K. STUDER, HILMER NEGRETE, PATRICIA A. CIvEN, and FREDERICK R. DERUBERTIS Department of Medicine, VA Medical Center, and University of Pittsburgh, Pittsburgh, Pennsylvania, USA

the mediation of renal dysfunction [8]. However, direct effects of TX on the mesangial cell (MC) have now been demonstrated that may contribute to the development of glomerulosclerosis. Bruggein mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we man et al found that TX increases human MC synthesis of type IV collagen, laminin, and fibronectin and the mRNA levels of these examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor /3 (TGF-/3) bioactivity, which in turn induces proteins [9, 10]. Mene et al similarly showed that TX stimulates enhanced matrix protein synthesis. In cultured rat MC, the TXAJ protein synthesis, including collagen type IV, in cultured human prostaglandin endoperoxide analogue U-46619, but not exogenous human MC [11]. Fibronectin (Fn) accumulation is also increased in rat platelet TGF-/31, activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 MC exposed to TX and a high ambient concentration of glucose and TGF-/31 stimulated fibronectin (Fn) synthesis in MC, as shown by [12]. The pathways through which TX induces changes in MC [35S]methionine incorporation into immunoprecipitable Fn. Pan-specific matrix protein synthesis are incompletely understood. MC have Protein kinase C signals thromboxane induced increases in fibronectin synthesis and TGF.p bioactivity in mesangial cells. Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis

rabbit anti-TGF-f3 antibody blocked the increases in Fn synthesis induced by exogenous TGF-/3 and those induced by U-46619 at 24 to 72 hours after

addition. Anti-TGF-/3 antibody did not block the small increases in Fn synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-/3. Anti-TGF-13 antibody also failed to block activation of PKC by U-46619. U-46619 and 50 n of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (latent plus active) TGF-/3 in MC culture media,

high affinity TX receptors [13, 14]. Upon binding to these

receptors, TX activates phospholipase C, stimulates increases in inositol phosphate turnover, elevates cytosolic calcium and diacylglycerol, and activates protein kinase C [14—17]. Recent studies in

our laboratory [17] have implicated rapid activation of protein kinase C (PKC) by TX as a signal for increased Fn synthesis in as assayed with the mink lung epithelial cell bioassay system. PKC MC. However, the pathway by which activation of PKC subseinhibition with bisindolylmaleimide GF 109203X (GFX) or down-regulaquently initiates an increase in Fn synthesis in MC is not known. tion of PKC in MC by prior exposure to a high concentration (0.5 j.tM) of phorbol myristate acetate (PMA) blocked increases in TGF-/3 bioactivity induced by either U-46619 or PDBu. PKC down-regulation in MC also

Transforming growth factor J3 (TGF-/3) is a potent stimulus of MC matrix protein synthesis and also inhibits degradation of these

blocked increases in Fn synthesis induced by U-46619. By contrast,

proteins [18, 19]. There is now strong evidence to support a

exogenous TGF-/3 stimulated Fn synthesis in both intact MC and in MC with down-regulated PKC. The findings indicate that activation of PKC by U-46619 signals an increase in TGF-j3 bioactivity, which in turn stimulates Fn synthesis in MC by processes not dependent on PKC.

pathogenetic role for TGF-j3 in glomerulosclerosis [20—26], in-

Thromboxane (TX) has been implicated in the pathogenesis of renal injury in experimental models of nephropathy, as well as in some human renal disorders [1—71. Thus, TX synthetase inhibitors ameloriate pathological changes in the kidneys of patients with

cyclosporine toxicity [3], and prevent increases in basement membrane protein mRNA levels in the kidneys of diabetic mice [7]. TX antagonists improve renal function in lupus nephritis [4], and in post-obstructive nephropathy [5], as well as preventing albuminuria and increases in glomerular matrix expansion in the streptozotocin diabetic rat [6]. Many earlier studies focused on the actions of TX as a platelet aggregator and vasoconstrictor in

Received for publication October 17, 1994 and in revised form February 15, 1995 Accepted for publication March 27, 1995

© 1995 by the International Society of Nephrology

cluding the demonstration in vivo that renal transfection with cDNA for TGF-/31 leads to the development of this histologic change [27]. In preliminary studies, we found evidence of a role for TGF-f3 in the expression of the action of TX to increase Fn synthesis in MC after 24 hours [28]. Thus, anti-TGF-f3 antibody blocked TX induced increases in Fn synthesis in MC, and TX increased TGF-13 bioactivity in MC culture media [28]. TGF-f3 has

similarly been implicated in the mediation of several other agents to increase MC matrix protein synthesis, including high ambient concentrations of glucose [29], low density lipoproteins [30] and angiotensin II [31]. Notably, all of these stimuli of matrix protein

synthesis activate PKC [30, 32, 33]. In the present study, we examined the possibility that TX activation of PKC is a signal to increase TGF-/3 bioactivity in MC, which in turn mediates the sustained increases in Fn synthesis observed in response to TX.

Methods Rat MC culture MC were cultured from collagenase treated glomerular cores obtained from the kidneys of 120 to 150 g female Sprague-Dawley

rats by graded sieving as previously reported [12]. They were maintained in RPMI-1640 medium supplemented with 15% fetal

422

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Studer et a!: Activation of PKC by U-46619

calf serum (FCS), 100 U/mi penicillin, 100 g/ml streptomycin, 5

PKC activity

g/ml insulin, 5 jg/m1 transferrin, and 5 nglml sodium selenite In situ PKC activity was determined as previously reported in (Sigma Chemical Co., St. Louis, MO, USA) at 37°C in 5% CO2 detail from this laboratory [12, 17]. Briefly, MC were grown to and 95% air. At confluence, MC were routinely rendered quies- confluence, media serum reduced to 1%, and the cells labeled cent by incubation in media with FCS reduced to 1% for 24 hours. with 32P-orthophosphate (New England Nuclear, Boston, MA, Test agents were then added. Cell passages 2 to 8 were used in USA) for four hours. MC were exposed to test agents as described these studies. in the figure legends and the acid soluble 80 kD myristoylated alanine rich C kinase substrate (MARCKS) protein extracted and Bioassay of TGF-J3 activity separated by electrophoresis. The PKC data are expressed as TGF-13 bioactivity in MC culture media was assayed using the mink lung epithelial cell (MLEC) assay with minor modifications of published procedures [34, 35], as previously reported from this laboratory [30]. Briefly, media were collected from MC cultured as described in the figure legends. Bovine serum albumin (BSA)

and protease inhibitors were added to final concentrations of 1

mg/ml BSA and 1 g/ml each of aprotinin, leupeptin, and

density units, calculated as the ratio of the phosphorylation of the

80 kD MARCKS protein to a 40 kD reference protein, whose phosphorylation did not change under the conditions studied. Materials

Materials were obtained from sources noted above, or as

pepstatin. The samples were centrifuged to remove cell debris and previously reported [30]. held at 4°C until assayed. Latent TGF-13 was activated by heating media to 80°C for 15 minutes. MLEC, American Type Culture

CCL-64, were seeded in MEM supplemented with 10% FCS, antibiotics, and glutamine. After six hours, medium FCS was reduced to 0.5% and the culture continued for 18 hours. Media from MC cultures or an equivalent volume of unconditioned MC media (RPMI-1640) containing exogenous TGF-131 standards were added to the MLEC. After 24 hours, the cells were pulse labeled with [3H]-thymidine (New England Nuclear, Boston, MA,

Statistics

Experiments were performed at least three times and data

shown as means SE. Each incubation condition was represented by duplicate or triplicate wells. Replicates from a single culture were averaged and entered as one value for purposes of statistical analysis. ANOVA was performed to compare multiple groups,

and the significance of differences between mean values was determined by Student's t-test.

USA) for two hours, washed with phosphate buffered saline

Results (PBS), and the incorporation of [3H]-thymidine into TCA precipFn synthesis in MC exposed to 1 jM of the TXA2/prostaglandin itable material determined by liquid scintillation counting. Standard curves for TGF-j31 bioactivity were prepared using exoge- endoperoxide analog U-46619 for 24 hours was approximately nous human TGF-131 (0.1 to 10 pM). Inhibition of MLEC [3H]- 2.5-fold higher than control (Fig. 1). As previously reported [17], thymidine ranged from 92% (10 M exogenous TGF-13) to 3% (0.1 this concentration of U-46619 induced maximal increases in Fn M TGF-13), with an IC50 of approximately 0.7 M under the assay synthesis at 24 hours. As is also shown in Figure 1, 1 M exogenous conditions used. Media samples were diluted to fall within the TGF-f3 also stimulated Fn synthesis in MC. The latter was the range of the standard curve. Unconditioned MC media containing lowest concentration of exogenous TGF-13 to increase Fn synthethe final concentrations of agonists and inhibitors present in sis after 24 hours significantly, although small and variable media from MC cultures were employed as controls in the increases were observed in response to 0.5 M TGF-p. Maximal bioassay system. The extent to which the action of culture media (up to 5-fold) increases in Fn synthesis were observed in response from MC to inhibit [3H]-thymidine incorporation in MLEC was to 50 pM TGF-f3 (not shown). Neither U-46619 nor TGF-13 attributable to TGF-13 bioactivity was examined by addition of 30 significantly altered MC number after 24 hours, and the increases g/ml of anti-TGF-13 antibody to the media samples prior to in Fn synthesis were evident whether expressed on the basis of cell bioassay. Anti-pan specific TGF-f3 antibody, but not control rabbit number (Fig. 1) or culture protein content (not shown). As is also IgG, completely abrogated the effects of MC culture media to shown in Figure 1, the addition of 30 g/ml pan-specific antisuppress [3H]-thymidine incorporation in MLEC, as previously TGF-/3 antibody to the cultures five minutes before U-46619 or TGF-13 blocked the increase in Fn synthesis induced by both reported [30]. agents. By contrast, the Fn synthetic response to U-46619 or Fn synthesis TGF-13 in the presence of 30 Wml control rabbit IgG (Fig. 1) did Fn synthesis was measured as [35S]-methionine ([35S]-Met) not differ from responses in the absence of IgG (not shown). incorporated into immunoprecipitable material extracted from Phosphorylation of the 80 kD MARCKS protein was increased the cells plus matrix after a four-hour pulse label as described in twofold 15 minutes after exposure of MC to U-46619, and the figure legends. MC were grown to confluence, FCS reduced to anti-TGF-/3 antibody had no effect on this response (Fig. 1). In 1%, and agonists added as described. After culture for the times contrast to U-46619, however, concentrations of exogenous indicated, the cells were washed twice with PBS and incubated in TGF-3 from 1 p (Fig. 1) to 50 M (not shown) failed to alter methionine-cysteine-deficient minimum essential media (MEM) MARCKS protein phosphorylation. with 25 j.tCi of [35S]-Met (Tran35S-label, ICN, Irvine, CA, USA).

[35S]-Met labeled Fn was immunoprecipitated with human Fn antiserum (Collaborative Research Inc., Bedford, MA, USA) and assayed as previously described [12]. Fn synthesis is expressed as dpm/103 cells or dpm/.tg protein, determined using bicinchoninic acid (Sigma Chemical Co.).

Figure 2 shows the effects of U-46619 and exogenous TGFj3 on FN synthesis in MC with intact PKC and in cells whose PKC had

been markedly downregulated by exposure to 0.5 LM phorbol myristate acetate (PMA) for 24 hours prior to the addition of U-46619 or exogenous TGF-p1. Sustained exposure of MC and other cells to this concentration of PMA decreases PKC activity to

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fibronectin synthesis in mesangial cells. Mesangial cells were grown to confluence as described in Figure 1. Where indicated, PKC was downregulated by exposure to 0.5 sM PMA for 24 hours; the DMSO vehicle (0.05%) for PMA was added where indicated. After 24 hours, media was changed, and the culture continued for 24 hours with PMA or vehicle and other test agents as indicated. Fibronectin synthesis was determined 24 hours after addition of TGF-13 or U-46619. Values are mean se (N = 4). < 0.05 versus Symbols are: (LI) control; () TGF3; (•), U-46619. < 0.05 versus corresponding U-46619 value in corresponding control. vehicle treated cells.

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of TGF-J3 in media by 145% over basal (0.53 M to 1.3 pM), and increased total (latent + active) TGF-/3 bioactivity by 43% (32 to 46 pM). Figure 3 also shows the effects on these TGF-13 bioactivities of bisindolylmaleimide GF 109203X (GFX), a potent and

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relatively specific inhibitor of PKC [361. We have previously shown that GFX blocks the Fn synthetic response to U-46619 in MC [17]. GFX did not alter basal accumulation of total TGF-f3 bioactivity in media, but suppressed the basal level of the active fraction to 30% of the value observed in cultures exposed to the GFX vehicle alone. By contrast, GFX completely blocked the increases in both active and total TGF-f3 bioactivity induced by U-46619.

Fig. 1. Effects of U-46619 and TGF-13 on mesangial cell PKC activity (B)

and fibronectin iynthesis (A) in the presence and absence of antibody to TFG-/3. Mesangial cells were grown to confluence in 15% FCS and medium changed to contain 1% FCS for 24 hours to growth arrest cells prior to addition of test agents. Eighty kD MARCKS phosphorylation was determined 15 minutes after the addition of 1 pivi TGF-/3, or 1 ILM U-46619

in the presence of 30 sWml of control rabbit IgG or pan-specific TGF-13 antibody. Fibronectin synthesis was determined 24 hours after addition of TGF-/3 or U-46619. Control IgG alone did not affect PKC activity or Fn synthesis in MC. Symbols are: (LI) control; () U-46619; (U) TGF-13. Values are means se (N = 4). *P < 0.05 versus corresponding control.

less than 2% of control values [12, 17, 36]. As shown in Figure 2,

PKC down-regulation had no significant effect on basal Fn

The role of PKC in the expression of the action of U-46619 to increase TGF-13 bioactivity in MC cultures was further examined in MC whose PKC was down-regulated by exposure to PMA for 24 hours prior to the addition of U-46619 (Fig. 4). U-46619 failed to increase TGF-13 bioactivity in media of MC whose PKC had been down-regulated. Consistent with the effects of GFX shown in Figure 3, PKC down-regulation had no effect on basal accumulation of total TGF-13. However, the basal level of the active fraction of TGF-13 in media was significantly reduced compared to that seen in media of control MC cultures. Figure 5 shows the effect of a low concentration (50 nM) of the PKC agonist phorbol dibutyrate (PDBu) on TGF-13 bioactivity in

media from MC. Low concentrations (50 to 100 nM) of PDBu synthesis. Moreover, in contrast to U-46619 which stimulated Fn have been shown to rapidly (15 mm) activate PKC in MC [12]. synthesis only in MC with intact PKC, exogenous TGF-p stimu- Despite a decline in the total PKC content of MC to 50 to 60% of lated Fn synthesis in MC with either intact or down-regulated initial values, PKC activation in MC in response to 50 to 100 flM PKC. PDBu is sustained for at least six days, as reflected by a persistent Figure 3 shows the effects of 1 sM U-46619 on TGF-/3 bioac- enhancement of MARCKS protein phosphorlyation [12]. As tivity in media of MC cultures. U-46619 increased the active form shown in Figure 5, 50 nM PDBu significantly increased the

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Fig. 3. Effects of U-46619 on TGF-p bioactivity in mesangial cell culture media in the presence and absence of the PKC inhibitor GF 109203X (GFX). (A) Active; (B) latent + active. Mesangial cells were grown to confluence

as described in Figure 1. GFX (2 gal), or the vehicle for GFX (0.05% DMSO) was added where indicated, followed 15 minutes later by the

addition of 1 gM U-46619. After 24 hours of culture, medium was harvested for determination of TUF-3 bioactivity. Total TGF-j3 bioactivity (latent plus active) was determined in media samples after heat activation.

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Fig. 4. Effects of PKC downregulation on basal and U-46619 mediated changes in TGF-f3 bioactivity in mesangial cell culture media. (A) Active; (B) active + latent. Mesangial cells were grown to confluence as described in Figure 1, and PMA (0.5 sM) or DMSO vehicle (0.05%) was added for 24 hours as indicated. Medium was then changed and 1 1kM U-46619 added

to some wells. Culture was continued for an additional 24 hours in the presence of PMA or its vehicle with or without U-46619, as indicated. The

conditioned media were harvested and assayed for TGF-!3 bioactivity. Values are mean SE (N = 4). p < 0.05 versus corresponding control. **p < 0.05 versus corresponding value in the vehicle treated cells.

concentration of active and total TGF-13 in media of MC cultured

in the presence of this agent for 24 hours. By contrast, in MC TGF-j3 was in the active form. In MC exposed to GFX for 24 subsequent addition of 50 nM PDBu failed to increase either hours, this value was reduced to 1.1%. After 48 hours exposure of active or total TGF-p bioactivity (Fig. 5). As noted above, PMA MC to 0.5 tM PMA, the active fraction of TGF-f3 in media was 1.5% of total activity. In MC cultured with U-46619, the fraction also reduced the basal levels of active but not total TGF-p. whose PKC had been down-regulated by prior exposure to PMA,

The preceding studies showed that inhibition of PKC decreased of total TGF-/3 in the active form was 5.6%. PDBu also increased and activation of PKC increased the fraction of total TGF-f3 in the the percentage of TGF-fJ in the active form to approximately 4% active state. In media of control MC cultures, 3.1% of the total (Fig. 5).

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mesangial cell culture media. Mesangial cells were treated as described in Figure 1 and media harvested at the times indicated for determination of TGF-/3 bioactivity. Values are mean SE (N = 3 to 6) (data for active and latent TGF-j3 at six hours have been multiplied by a factor of 2). Symbols are: (0) control; () U-46619. < 0.05 versus corresponding control.

Figure 6 shows the time course of U-46619 action on accumu5; U-46619 + IgG, 85 7; U-46619 + anti-TGF-j31, 82 8). lation of TGF-p bioactivity in MC culture media. The earliest The dose of anti-TGF-131 antibody employed in these studies was time point after addition at which U-46619 induced a significant sufficient to block the 50% increase in Fn synthesis induced in MC increase in either active or total TGF-p was six hours. Compared exposed to 25 M exogenous TGF-13 for six hours. By contrast, 2 to corresponding control values, U-46619 also increased accumu- LM GFX blocked U-46619 induced increases in both TGF-j3 lation of active and total TGF-J3 at 24, 48, and 72 hours. bioactivity and Fn synthesis at six hours and at the later time Concurrent increases in Fn synthesis occurred over this period, as points. previously reported [17]. However, while anti-TGF-j3 antibody (30 pg/mI) blocked increases in Fn synthesis induced by U-46619 from

Discussion 24 to 72 hours, it did not alter the 40% increase in Fn synthesis Previous studies have indicated that the action of TX to observed at six hours (IgG, 62 5 dpm/103 cells; anti-TGF-/3, 64 stimulate Fn synthesis by MC is signalled by activation of PKC

Studer et a!: Activation of PKC by U-46619

427

[17] and is dependent on TGF-f3 bioactivity [28]. The current affinity binding of TGF-/3 in MC was saturated at a concentration findings are consistent with the hypothesis that activation of PKC of between 10 and 15 p, with a kD of 5 M [42]. The concentraby TX in MC signals an increase in TGF-p bioactivity, which in tion response relationship observed between TGF-f3 and fibronec-

turn mediates the increases in Fn synthesis that occur in MC

tin synthesis is therefore consistent with the reported kD for

exposed to TX for 24 hours or longer. Thus, anti-TGF-J3 antibody TGF-p in MC. Although the basal levels of active TGF-J3 suppressed increases in Fn synthesis induced by either exogenous observed in the present study are below those previously reported TGF-13 or culture of MC with U-46619 for 24 hours or longer. By in culture media of MC, the increases in active TGF-)3 (0.53 to 1.3

contrast, anti-TGF- antibody did not alter the U-46619 activa- p, Fig. 3) induced by 24 hours of exposure to U-46619 appear tion of PKC. Accordingly, TGF-j3 antibody did not impair the sufficient to mediate the action of U-46619 on Fn synthesis, at this initial steps of the TX signalling pathway, specifically the TX- time, in view of the sensitivity of this parameter to a concentration receptor interaction and activation of PKC which have previously of exogenous TGF-!3 as low as 1 p. The low basal levels of active been linked to expression of TX effects on Fn synthesis in MC TGF-j3 in the control MC which had been rendered quiescent by [17]. Moreover, U-46619 and the PKC agonist PDBu significantly prior reduction in the concentration of FCS in the culture media increased both active and total TGF-13 bioactivity in media of probably did not modulate Fn synthesis in the control cells. This cultured MC. GFX, a potent inhibitor of PKC [37], blocked is implied by: (a) the failure of PKC inhibition or down-regulation U-46619 induced increases in these bioactivities. These findings to alter basal rates of Fn synthesis despite a concurrent reduction all support a role for PKC in transduction of U-46619 effects on in active TGF-; and (b) the absolute concentration of active TGF-/3 bioactivity. Although the IC50 of GFX for PKA and TGF-f3 in media under control conditions (0.6 M or less), which phosphorylase kinase are 200 and 70 times greater, respectively, was below the minimal concentration of exogenous TGF-p (1 pM) than that for PKC [371, the inhibitory action of GFX is not entirely effective in stimulating Fn synthesis. selective for PKC relative to other kinases. Accordingly, the In contrast to the inhibitory action of anti-TGF-13 antibody on influence of down-regulation of PKC in MC was also examined to increases in Fn synthesis observed in MC exposed to TX for 24 assess further the role of PKC in expression of U-46619 stimu- hours or longer, anti-TGF-j3 antibody failed to block the 40% lated increases in TGF-13 bioactivity. As shown in Figure 2 and increases in Fn synthesis after only six hours of culture with TX. previously reported [17], down-regulation of PKC by prior expo- Moreover, the small increases in the active fraction of TGF-f3 sure of MC to PMA blocked the action of U-46619 to increase Fn (<0.5 pM) observed after this brief exposure to U-46619 were synthesis. The present data demonstrate that PKC down-regula- likely inadequate to account for the corresponding early increases

tion also prevented the increases in TGF-13 bioactivity in MC culture media induced by both U-46619 and PDBu. In contrast,

down-regulation of PKC failed to alter the increases in Fn synthesis induced in MC by exogenous TGF-p. These latter findings, and the failure of exogenous TGF-p to activate PKC in MC (Fig. 1), suggest that the action of TGF-/3 on Fn synthesis in these cells is not dependent on PKC. However, since not all PKC isozymes are down-regulated by phorbol ester [38], it is possible that PKC isoforms such as PKC-&, which is resistant to down-

in Fn synthesis, since 25 M exogenous TGF-p yielded only

modest increases in Fn synthesis in this time frame. In our earlier studies [17], we demonstrated that inhibition or down-regulation of PKC activity prevented both the early (6 hr) and later increase

in Fn synthesis induced by TX. In the current study, PKC inhibition with GFX similarly blocked both the early and subse-

quent increases in TGF-(3 induced by TX. Nevertheless, the failure of antibody to TGF-p to block the acute effects of TX on Fn synthesis suggest that this action is not dependent on TGF-13.

regulation, could be involved in the expression of actions of In this regard, serum, a potent activator of PKC [12], rapidly TGF-13 on Fn synthesis.

The concentrations of both active and latent TGF-j3 found in MC culture media in the current studies are low relative to those previously reported by Kaname et a! [35] and Kagami et al [31]. With respect to the active fraction, these groups reported basal media concentrations of TGF-j3 of 14 M after 24 hours [35] and 10 M after 48 hours culture of MC [31]. The fraction of active TGF-p constituted 4% [35] and 12% [31] of total TGF-f3 bioactivity under basal MC incubation conditions, compared to 3.1% in the present study. Whether these differences are a function of the

MC culture conditions, the processing of the media prior to bioassay (addition of protease inhibitors, dialysis of samples) or other factors is not certain. The concentrations of TGF-p detected in MC culture media in the present study are also much lower than the concentrations of exogenous TGF-13 often used [39—42] to stimulate increases in mRNA levels for Fn (40 to 800 pM) or

protein and collagen synthesis (10 to 120 pM) in MC. In the current study, increases in Fn synthesis by MC were observed in response to concentrations of TGF-p1 as low as 1 M (Fig. 1), with maximal stimulation in response to 50 p. The responsiveness of Fn synthesis to TGF-13 in MC thus approximates that of collagenase sensitive proline incorporation which was maximally stimulated by 10 M TGF-p [42]. MacKay et al also found that high

increases Fn gene expression [43]. Similarly, Naham, Rovin and Leonhart [44] reported increases in Fn mRNA in MC 30 minutes after exposure to phorbol ester, with subsequent increases in both

matrix and medium Fn content by four hours. These rapid responses and the acute increases in Fn synthesis induced by TX may reflect genomic effects of PKC expressed independent of TGF-j3. A number of prior observations have implicated PKC as a signal

for increased TGF-13 synthesis and/or secretion. Thus, Akhurst, Fee and Balmain reported phorbol ester induced TGF-$3 mRNA in epidermal cells [45]. Moreover, regions in the TGF-13 gene that are responsive to phorbol ester have been identified [46]. PKC has also been implicated in increases in TGF-p mRNA [33, 35] and TGF-13 bioactivity [47] observed in vascular smooth muscle cells. In MC, several activatOrs of PKC in addition to phorbol ester and U-46619 have now been shown to increase TGF-3 mRNA levels, bioactivity, or both. These include high concentrations of glucose [29], angiotensin II [31], and low density lipoproteins [30], all of which also increase MC matrix protein synthesis. Ziyadeh et a! [29] found that high concentrations of glucose, which activate PKC via de novo synthesis of diacyiglycerol [48], stimulate increased TGF-13 mRNA by murine MC. Kagami et al [31] showed that angiotensin II, another known agonist of PKC [49], increased

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Studer et a!: Activation of PKC by U-46619

All

TX

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+

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TGF-/3 mRNA levels in rat MC and TGF-f3 bioactivity in MC culture media. We recently found that exposure of rat MC to low density lipoproteins activated PKC and increased media TGF-/3 bioactivity [30]. Analogous to the finding with U-46619 in the present study, inhibition or down-regulation of PKC activity in MC prevented the increases in TGF-13 bioactivity induced by low density lipoproteins [30]. By contrast, our findings do not implicate PKC isoforms that are down-regulated by phorbol esters or inhibited by GFX in the pathway by which TGF-/3 stimulates Fn synthesis in MC. Thus, exogenous TGF-13 failed to activate PKC (Fig. 1), and downregulation of PKC in MC (Fig. 2) or the PKC inhibitor GFX (data not shown) failed to alter the increases in Fn synthesis induced by exogenous TGF-/3. There are data both supporting and rejecting a role for PKC activation in the mediation of TGF-13 effects on cell function. Muldoon, Rodland and Magun [50] reported that

prolonged treatment of fibroblasts with TGF-p stimulated calcium influx and phosphatidylinositol turnover, while Markovac and Goldstein [51] found that TGF-f3 caused a redistribution of PKC from the cytosolic to particulate fraction in microvessels of rat brains. More recently, Bradham et a! [52] concluded that

Fig. 7. Proposed pathway by which activators of PKC may contribute to mesangial matrix &pansion via increases in TGF-f3. See text for explanation.

activation of PKC was necessary for TGF-p induced downregulation of collagen II expression in chondrocytes. However, this was based on studies utilizing the serine/threonine protein kinase inhibitor H-7. Ohtsuki and Massague [531 had previously shown that H-7 inhibits responses to TGF-13 by interfering with a phosphorylation step unrelated to PKC. Results of two recent studies are consistent with our findings in MC. Anderson et a! [54] reported that TGF-/3 did not activate PKC in either LLC-PK1 or

MDCK renal epithelia! cell lines, and Mu, Ware and Kent [55] found analogous results in vascular smooth muscle cells derived from human saphenous veins. Thus, TGF-j3 may activate PKC in some cells, but this action is not apparent in MC and other renal cell lines. As noted above, increases in TGF-f3 mRNA, TGF-j3 bioactivity,

or both have been found in response to activators of PKC, including phorbol ester, glucose, low density lipoproteins, and angiotensin II [29—31, 33, 35, 42, 43]. In the present study, U-46619 and phorbol ester increased latent TGF-f3 and this action was also linked to activation of PKC. In view of earlier reported changes in mRNA levels for TGF-13, the increases in latent TGF-/3

bioactivity observed in response to U-46619 and phorbol ester in

429

Studer et al: Activation of PKC by (1-46619

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