JOURNAL OF CELLULAR PHYSIOLOGY 150:552-558 (1992)

Shear-Induced Platelet-Derived Growth Factor Gene Expression in Human Endothelial Cells Is Mediated by Protein Kinase C HSYUE-JENHSIEH, NAN-QIAN 11, AND JOHNA. FRANCOS* Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802 Our previous studies have shown that steady shear stress causes a transient increase of platelet-derived growth factor (PDGF) A and B chain mRNA levels in human umbilical vein endothelial cells (HUVEC). In the present study, we elucidated the signaling pathway of shear stress in HUVEC by examining the roles of protein kineses, intracellular calcium, cyclooxygenase, and guanine nucleotidebinding proteins (G proteins) in the PDGF gene induction by shear. The protein kinase C inhibitors, H 7 and staurosporine, strongly inhibited the shear-induced PDGF gene expression in HUVEC. In contrast, HA1004, a CAMP- and cGMPdependent protein kineses inhibitor, was only slightly inhibitory. BAPTA/AM, an intracellular calcium chelator, partially (50%) inhibited the shear-induced PDGF gene expression. The cyclooxygenase inhibitors, ibuprofen and indomethacin, were slightly inhibitory. A 35-50% inhibition of shear-induced PDGF gene expression was found with GDP-P-S, an inhibitor of G proteins. These results suggest that shear-induced PDGF gene expression in HUVEC is mainly mediated by protein kinase C activation and requires intracellular calcium. Furthermore, G proteins seem to be involved in this process, whereas prostaglandin synthesis via cyclooxygenase pathway is not. We propose a mechanism of shear-induced PDGF gene expression in HUVEC: Shear stress, either directly or indirectly (G protein-mediated), enhances the membrane phosphoinositide turnover via phospholipase C, producing diacylglycerol, an activator of protein kinase C. The activated protein kinase C then triggers the subsequent PDGF gene expression.

It has been shown that wall shear stress in the vascu- shearing flow (Hsieh et al., 1991). Moreover, the peak lar system is maintained a t a n optimal level by a n levels of PDGF A and B mRNA were also shear-depenautoregulatory mechanism (Kamiya and Togawa, dent (Hsieh et al., 1991). These observations suggest 1980; Zarins et al., 1987). Normal blood vessels can that PDGF may be involved in the adaptation of blood respond to shear stress and adapt their blood vessel vessels to flow mediated by the endothelium. The mechanisms for the shear-induced PDGF gene diameter at two levels: acutely through vasoactive mechanisms (flow-dependent constriction or dilation), expression, however, are not well understood. Previous and chronically by adjusting vascular caliber (Kamiya studies have shown that shear stress can stimulate and Togawa, 1980; Zarins et al., 1987). The endothe- membrane phosphoinositide turnover (perhaps via lium, which lines the inner surface of blood vessels and phospholipase C) in endothelial cells, producing the is in direct contact with blood flow, appears to be essen- second messengers inositol trisphosphate UP,) and diatial in this autoregulatory mechanism (Langille and cylglycerol (DAG) (Bhagyalakshmi and Frangos, ODonnell, 1986). In the normal response of blood ves- 1989a,b; Nollert et al., 1990; Prasad et al., 1989). IP, sels to flow, i t is generally believed that the roles of the triggers the release of calcium from intracellular pools, endothelium are to sense blood flow and mediate any and DAG is a n activator of protein kinase C, a major kinase responsible for many cellular responses, e.g., necessary response of the smooth muscle cells (SMC). PDGF, a mitogen for SMC and a vasoconstrictor gene expression, cell proliferation, etc. (Nishizuka, (Berk e t al., 19861, is believed to be involved in this 1986). The induction of PDGF gene expression by shear process, especially in the chronic change of vascular stress, therefore, may be mediated by protein kinase C caliber, which involves remodeling and/or growth of activation. In addition, shear increases prostaglandin SMC and connective tissues. Endothelial cells can se- I, synthesis, which may have a n autocrine action on the crete PDGF, which may bind to PDGF receptors on underlying SMC in vivo (Ross et al., 1986). Our previous results showed that steady shear stress induced a transient increase in PDGF A and B mRNAs levels in human umbilical vein endothelial cells (HU- Received July 30,1991; accepted October 7,1991. VEC), with peaks occurring 1.5-2 h after the onset of *To whom reprint requestskorrespondence should be addressed. 0 1992 WILEY-LISS, INC.

SHEAR EFFECTS AND PROTEIN KINASE C

intracellular CAMPlevels in endothelial cells (Frangos et al., 1985; Reich e t al., 1990). Recently, guanine nucleotide-binding proteins (G proteins) have been demonstrated to be responsible for the regulation of phospholipase C in agonist-stimulated endothelial cells (Voyno-Yasenetskaya e t al., 1989). In order to clarify the signaling pathways of the induction of gene expression by shear stress in endothelial cells, we have examined the roles of protein kinases, intracellular calcium, cyclooxygenase, and G proteins in the PDGF gene induction by shear stress. Our results show that shear-induced PDGF gene expression is mediated by protein kinase C activation, which is calciumiphospholipid dependent. Furthermore, G proteins appear to be involved in this process, whereas prostaglandin synthesis and CAMP- and cGMP-dependent protein kinases are not.

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ski and Sacchi, 1987). After quantification by measuring absorbance at 260 nm, 25 to 30 pg of RNA samples (same amount per lane for each gel) were electrophoresed on 1.2% MOPS-formaldehyde agarose gels (Ausubel et al., 19871, transferred to nylon membranes (Nytran 0.45 pm, Schleicher & Schuell Inc., Keene, NH) (Ausubel et al., 19871, and fixed by UV irradiation (user’s manual for Schleicher & Schuell Nytran membrane). The 32P-labelled cDNA fragments (Multiprime DNA labelling system, Amersham, Arlington Heights, IL) (3,000 Ci/mmol, Amersham) were used as probes. A 1.3 kb-EcoR I fragment containing PDGF A cDNA was isolated from plasmid PDGF-A-13.1 (received as a gift from C. Betsholtz) (Betsholtz et al., 1986).A 1.48 kb-Pst I/BamH I fragment containing PDGF B cDNA was isolated from plasmid pGGS2.7dBH (received as a gift from C. D. Rao) (Rao et al., 1986). A 0.96 kb-Hinc II/Bgl I fragment containing GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA was isolated from plasmid pHcGAP (deposited by R. Wu, received from American Type Culture Collection, Rockville, MD) (Tso et al., 1985). Membranes were hybridized with PDGF A probe first. After washes, membranes were exposed to Kodak XAR films a t - 70°C for autoradiography (Ausubel et al., 1987). Then the bound probe was stripped off. The same membrane was rehybridized with PDGF B probe and then with GAPDH probe (user’s manual for Schleicher & Schuell Nytran membrane).

MATERIALS AND METHODS Cell culture and flow loop experiments Primary cultures of HUVEC were harvested from human umbilical veins by collagenase treatment and plated on glass slides (75 x 38 mm) precoated with 0.2% gelatin (Sigma Chemicals, St. Louis, MO), as described elsewhere (Frangos et al., 1985). Cells were grown to confluence within 3 4 days in Medium 199 containing 20% fetal bovine serum (Hyclone Laboratories, Logan, UT), 1% penicillin-streptomycin, and 1% glutamine (Sigma). One day before using the cultures Quantification of hybridization signals in experiments, the medium was changed. Cells were sub’ected to well-defined steady shear stress (16 dynes/ The intensity of each hybridization band on XAR . a parallel plate flow chamber for 1.5 h , where film was determined by optical densitometry (QUICK cmd) in medium was circulated and driven by a constant-head SCAN JR. & QUICK QUANT 111,Helena Laboratories, flow loop (Frangos e t al., 1988). To eliminate possible Beaumont, TX). Each GAPDH (glyceraldehyde-3-phoseffects due to the addition of fresh medium, each flow phate dehydrogenase) band served a s the internal stanloop was primed with the same medium in which the dard to normalize PDGF A and B signals since GAPDH cells were previously incubated. To investigate the is constitutively expressed in HUVEC. mechanism of shear-induced PDGF gene expression in RESULTS HUVEC, the following inhibitors were added sepaEffect of HA1004 and H7 on PDGF rately to the media l h before the flow experiments: mRNA levels protein kinase C inhibitors l-(5-isoquinoline-sulfonyl)2-methylpiperazine (H7) (20 pM final concentration) To study the roles of protein kinases in shear-induced (Hidaka et al., 1984) (Seikagaku America, Rockville, PDGF gene expression, HUVEC were exposed to steady MD) or staurosporine (0.1 pM) (Tamaoki et al., 1986) shear stress (16 dynes/cm2) for 1.5 h in the absence or (Sigma); CAMP-and cGMP-dependent kinase inhibitor presence or HA1004, a CAMP- and cGMP-dependent N-(2-guanidinoethyl)-5-isoquinolinesulfonamidehy- kinases inhibitor. Similar experiments were also cardrochloride (HA1004) (20 pM) (Hidaka e t al., 1984) ried out with H7, a protein kinase C inhibitor. North(Seikagaku America); membrane-permanent calcium ern analysis showed that shear-induced elevation of chelator bis-(0-aminophenoxy)-ethane-N,N,N’,N’-tetPDGF A and B mRNA levels in HUVEC was slightly raacetic acid,tetra(acetoxymethyl)-ester (BAPTNAM) inhibited by HA1004 and almost totally abolished by (2.5 pM) (Tsien, 1980) (Calbiochem, San Diego, CAI; H7 (Fig. lA,B). In contrast, GAPDH mRNA levels, as cyclooxygenase inhibitors ibuprofen (20 pM) (Sigma) or a n internal standard, remained relatively constant, inindomethacin (14 pM) (Oliw, 1980) (Sigma); phospholi- dicating that H7 selectively inhibited the shear-inpase C inhibitor neomycin (0.3 or 1.0 mM) (Schacht, duced PDGF gene expression (Fig. 1C). Relative levels of PDGF A and B mRNA were deter1976) (Sigma); G protein inhibitor guanosine 5‘-0-(2thio) diphosphate (GDP-P-S) (100 p M ) (Eckstein et al., mined by densitometric scanning of each hybridization band. After normalized to GAPDH mRNA, we found a 1979) (Sigma). 93% reduction in shear-induced PDGF A mRNA level RNA isolation and Northern blot analysis by H7 and only 15% reduction by HA1004 (Fig. 2A). Immediately after being subjected to shear for 1.5 h, Similar inhibitory effects on shear-induced PDGF B cells were lysed in 4 M guanidinium thiocyanate solu- mRNA levels were also observed (Fig. 2B). H7 and tion (Chomczynski and Sacchi, 1987). Total RNA was HA1004 have similar inhibitory effects on CAMP-and isolated from the cell lysate by phenol-chloroform ex- cGMP-dependent protein kinases, but H7 is a more potraction followed by ethanol precipitation (Chomczyn- tent inhibitor of protein kinase C than HA1004 (K, of

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Fig, 1. Effect of HA1004 and H7 on PDGF mRNA levels. HUVEC were either in static condition (Stationary)or exposed to steady shear stress (16 dynesicm’) for 1.5 h (Flow) in the absence (Control), or presence of HA1004 (20 r M ) or H7 (20 KM). Total RNA was isolated from cells for Northern analysis of PDGF A (A), PDGF B (B), and GAPDH mRNA (C). Each lane represents the combined RNA sample from two experiments performed under the same condition. Shearinduced elevation of PDGF A and B mRNA levels in HUVEC was slightly inhibited by HA1004 but almost totally abolished by H7. GAPDH mRNA levels remained relatively constant. See text for abbreviations.

H7 for protein kinase C is 6 pM; Ki of HA1004 for protein kinase C is 40 pM) (Hidaka et al., 1984). Therefore, our results show that protein kinase C rather than other kinases is a major mediator for the shear-induced PDGF gene expression in HUVEC. Moreover, the levels of PDGF mRNA in the stationary controls were also reduced by H7, indicating that not only shear-induced but also basal PDGF gene expression are dependent on protein kinase C activity.

Effect of BAPTA/AM, ibuprofen, indomethacin, and staurosporine on PDGF mRNA levels To confirm the role of protein kinase C, and to study the roles of intracellular calcium and cyclooxygenase in shear-induced PDGF gene expression, HUVEC were subjected to steady shear stress (16 dynesIcm2) for 1.5 h in the absence or presence of staurosporin, a potent protein kinase C inhibitor. Similar experiments were carried out with BAPTAIAM, a n intracellular calcium

HA1004

H7

Fig. 2. Normalized effect of HA1004 and H7 on PDGF mRNA levels. Intensities of PDGF A and B mRNA signals in Figure 1were normalized by GAPDH mRNA. A and B show relative PDGF A and PDGF B mRNA levels in HUVEC under stationary (m)or flow. ( 1 condition in the absence (Control), or presence of HA1004 or H7. HA1004 reduced PDGF A and B mRNA levels in HUVEC subjected to flow by 15%, whereas H7 reduced PDGF A and B mRNA levels by 93% and 73%, respectively.

chelator, also with ibuprofen and indomethacin, both as cyclooxygenase inhibitors. Northern analysis demonstrated that shear-induced elevation of PDGF A and B mRNA levels in HUVEC was almost completely blocked by staurosporine, partially inhibited by BAPTAIAM, but only slightly inhibited by ibuprofen and indomethacin (Fig. 3A,B). GAPDH mRNA levels remained relatively constant, indicating that staurosporin and BAPTAIAM selectively inhibited the shearinduced PDGF gene expression (Fig. 3C). Relative levels of PDGF A and B mRNA were measured by densitometry, then normalized by the respective GAPDH mRNA level (Figs. 4,5). There was a 90% reduction in shear-induced PDGF A mRNA level by staurosporin and a 50% reduction by BAPTAIAM (Fig. 4A). The reductions in shear-induced PDGF A mRNA level by ibuprofen and indomethacin were only 10% and 20%, respectively (Fig. 5A). Similar inhibitory effects on shear-induced PDGF B mRNA levels were also observed (Fig. 5B). The levels of PDGF mRNA in stationary controls were also reduced by staurosporine, indicating that not only shear-induced but also basal PDGF gene expression were dependent on protein kinase C activity. These results demonstrated that protein kinase C activation and intracellular calcium both were required in the shear-induced PDGF gene expres-

SHEAR EFFECTS AND PROTEIN KINASE C

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Fig. 3. Effect of BAPTAIAM, ibuprofen, indomethacin, and staurosporine on PDGF mRNA levels. HUVEC were either in static condition (Stationary) or exposed to steady shear stress (16 dynesicm') for 1.5 h (Flow) in the absence (Control), or presence of BAPTA/AM (2.5 pM), ibuprofen (20 pM), indomethacin (14 pM) or staurosporine (0.1 pM). Total RNA was isolated from cells for Northern analysis of PDGF A (A),PDGF B (B),and GAPDH mRNA (C). Each lane represents the combined RNA sample from two experiments performed under the same condition. Shear-induced elevation of PDGF A and B mRNA levels in HUVEC was partially inhibited by BAPTAIAM, almost completely blocked by staurosporine, but only slightly inhibited by ibuprofen and indomethacin. GAPDH mRNA levels remained relatively constant.

sion in HUVEC, whereas cyclooxygenase activity for prostaglandin synthesis was not important.

Effect of neomycin and GDP-P-S on PDGF mRNA levels To study the roles of G proteins and phospholipase C in shear-induced PDGF gene expression, HUVEC were exposed to steady shear stress (16 dyneslcm') for 1.5 h in the absence or presence of GDP-P-S, a n inhibitor of G proteins. Similar experiments were carried out with neomycin, a phospholipase C inhibitor. Northern analysis showed that shear-induced PDGF gene expression in HUVEC was reduced by GDP-P-S (Fig. 6A,B), but GAPDH mRNA levels remained relatively constant (Fig. 6C), indicating that GDP-P-S selectively reduced the shear-induced PDGF gene expression. Neomycin, however, reduced GAPDH as well as PDGF mRNA levels in a dose-dependent manner (Fig. 6A,B,C).

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Fig. 4. Normalized effect of BAPTNAM and staurosporine on PDGF mRNA levels. Intensities of PDGF A and B mRNA signals in Figure 3 were normalized by GAPDH mRNA. A and B show relative PDGF A and PDGF B mRNA levels in HUVEC under stationary (0) or flow ( W ) condition in the absence (Control), or presence of BAPTA/AM or staurosporine. BAPTAiAM reduced PDGF A and B mRNA levels in HUVEC subjected to flow by 50%, whereas staurosporine inhibited PDGF A and B mRNA levels by 90%and 80%, respectively.

Relative PDGF mRNA levels were determined by densitometry. There was a 35% reduction in shear-induced PDGF A and 50% reduction in PDGF B mRNA levels by GDP-P-S (Fig. 7A,B), indicating that G proteins appeared to be involved in the shear-induced PDGF gene expression. However, the reduction in shear-induced PDGF mRNA levels by neomycin was not conclusive, since neomycin reduced both PDGF and GAPDH mRNA levels (the latter being constitutively expressed). Neomycin has been widely used as a phospholipase C inhibitor (Schacht, 1976), but according to these results it appears to have nonspecific inhibitory effects on gene transcriptions. Therefore, great care must be taken in using neomycin as a phospholipase C inhibitor.

DISCUSSION Several studies have shown that shear stress stimulates phosphoinositide turnover in endothelial cells, producing the second messengers IP, and DAG, possibly via phospholipase C (Bhagyalakshmi and Frangos, 1989a,b; Nollert et al., 1990; Prasad et al., 1989). IP, is believed to activate the release of calcium from intracellular pools, whereas DAG is a n activator of protein kinase C (Nishizuka, 1986). Since fairly high activity of protein kinase C and a large number of protein substrates have been found in endothelial cells (Mackie

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