Regulation of Genetic Expression in Shear Stress-s timul ated En dothelia 1 Cells” M. U. NOLLERT? N. J. PANARO, AND L. V. McINTIREC Cox Laboratory of Biomedical Engineering Institute of Biosciences and Bioengineering Rice University Houston. Texas 77251-1892 INTRODUCTION

The endothelial cell monolayer lining the vasculature plays an active role in modulating a number of important physiological processes including regulation of vascular tone and participation in the reactions involved in thrombosis and fibrinolysis. By the rapid synthesis and release of bioactive compounds such as prostacyclin and endothelium-derived relaxing factor (EDRF),’ the endothelium can respond immediately to an external stimulus. Over a period of several hours, the cells respond by altering the synthesis and release of proteins such as tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), endothelin (ET), fibronectin (FN), von Willebrand’s factor (vWF), basic fibroblast growth factor (bFGF), and transforming growth factor P l (TGF-Pl). Wez4 and others5s6have demonstrated that the mechanical forces caused by flowing blood lead to alterations in protein synthesis and genetic expression. The mechanisms by which these cells detect and respond to shear stress and cyclical strain caused by blood flow are not well understood. One of the most rapid responses of the cell to an external stimulus, such as the binding of an agonist to its receptor or, perhaps, mechanical stress, is the generation of intracellular signaling molecules. Examples of important second messenger molecules are cyclic adenosine monophosphate (CAMP),’ inositol-1,4,5-trisphosphate ( I n ~ 1 , 4 , 5 P ~and ) , ~calcium ions (Ca2+).Elevated levels of these compounds can directly activate some metabolic pathways as well as stimulate the activity of certain protein kinases. The protein kinases in turn modulate the activity of other enzymes, eventually leading to altered genetic expression. The mechanism for movement of information from the cytosol to the nucleus is still poorly understood. The selective induction of so-called “immediate early genes” (IEGs), which are transcribed within minutes after the cell is stimulated, plays a critical role in regulating the transcription of other proteins. These genes code for proteins that bind to DNA and may play an aThis work was partially supported by Grant Nos. HL 18672 and NS 23327 from the National Institutes of Health, Grant No. C-938 from the Robert A. Welch Foundation, and Grant No. 003604048 from the Texas Advanced Technology Program. bCurrent address: School of Chemical Engineering and Materials Science, University of Oklahoma, Norman, Oklahoma 73019. ‘To whom all correspondence should be addressed. 94

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important role in the signal transduction system that leads to altered protein synthesis within several hours after the initiation of shear stress. Consequently, studying the very rapid alterations in genetic expression of IEGs that occur after the initiation of flow may lead to a better understanding of the mechanism of shear stress signal transduction in endothelial cells.

SIGNAL TRANSDUCTION cAMP and CAMP-dependentKinase The receptors for several hormonal agents are linked to the enzyme, adenylyl cyclase, through a g-protein9 (FIGURE1). Adenylyl cyclase converts adenosine trisphosphate (ATP) into adenosine 3'3'-monophosphate (CAMP). Inside the cell, cAMP interacts with a variety of enzymes to modulate specific metabolic pathways including a number of protein kinases (PKA). In endothelial cells, elevated levels of cAMP have been associated with reduced production of PGI2 and EDRF,Io decreased permeability through the gap junctions,lI and increased lymphocyte adhesion and penetration through endothelial cell monolayers.I2 There is some evidence that mechanical stress can affect adenylyl cyclase activity. In a mouse lymphoma cell line, Watson13showed that hyposmolar swelling of the cell resulted in stimulation of adenylyl cyclase. Reich et al. l 4 demonstrated that primary human umbilical vein endothelial cells have an elevated level of intracellular cAMP after 15 minutes of exposure to a shear stress of 4 dyn/cm2. However, this increase was abolished in the presence of ibuprofen, suggesting that the change in CAMP levels is mediated by an arachidonic acid metabolite, possibly PG12. I P , Calcium, and Protein Kinase C A second receptor-mediated signal transduction system in endothelial cells is the pathway resulting in elevated levels of inositol-1,4,5-trisphosphate (Ins1,4,5P3). A number of different agonists affect these cells through this pathway including histamine, thrombin, bradykinin, and ATP. The specific receptors are linked to a g-protein that activates a phosphatidylinositol-specificphospholipase C, as is shown in FIGURE1. This enzyme breaks down phosphatidylinositol-4,5-bisphosphate into Ins1,4,5P3 and diacylglycerol.8 Elevated levels of diacylglycerol in the plasma membrane can cause translocation and activation of protein kinase C. Ins1,4,5P3 is soluble in the cytosol where it binds to receptors on the endoplasmic reticulum, causing release of calcium into the cytosol from internal stores. Locally elevated levels of calcium ion concentration are also required for enhanced protein kinase C activity. The evidence for the activation of this second messenger system in shear stress-stimulated endothelial cells comes from several different types of experiments. There is increased uptake of arachidonic acid into diacylglycerol and phosphatidylinositol in endothelial cells exposed to arterial levels of shear stress.l5 Also, the flow-stimulated production of PGI2 is reduced in the presence of the diacylglycerol lipase inhibitor, RHC-80267.I6 Both of these studies suggest that there is enhanced turnover of diacylglycerol, which may be the result of a phosphatidylinositol-

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specific phospholipase C. We have shown that there is a greater than twofold increase in the Ins1,4,5P3 levels within one minute of the initiation of flow." However, this increase may be mediated by ATP in the media because, in other studies, we18 and other^^^^^^ have shown that there is no increase in the cytosolic calcium level in cells exposed to step increases in flow with media that does not Plasma membrane

membrane hyperpolarization

phosphorylated

FIGURE 1. Signal transduction systems in endothelial cells that lead to altered genetic expression. Three of the many possible second messenger generation pathways that may be active in stimulated endothelial cells are shown. First, there is a potassium channel that is regulated by a g-protein. Opening of this channel causes hyperpolarization that may activate some membrane potential-sensitive pathways. Second, there is the receptor-mediated pathway that activates phospholipase C (PLC), causing the breakdown of phosphatidylinositol-4,5bisphosphate (PIP2) and the production of inositol-1,4,5-trisphosphate(IP3) and diacylglycerol (DAG). Third, there is the receptor-mediated pathway leading to activation of adenylyl cyclase and the formation of adenosine 3',5'-monophosphate (CAMP).

contain ATP. Only with micromolar quantities of ATP in the perfusing media do endothelial cells respond to increases in the flow rate by increasing their cytosolic calcium levels, at least as detected by the fluorescent dye, fura-2. The onset of flow greatly increases the convective transport of ATP to the cells and the cells respond to this increase in the local concentration of agonist.18 There is still the possibility that

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shear stress may increase local calcium concentrations in the vicinity of the plasma membrane, which is not detected by a cytosolic dye such as fura-2.

Membrane Hyperpolarization

One other possible signal transduction mechanism involves selective modulation of the permeability of the membrane to various ions. Lansman et ul.*’ described a stretch-activated nonselective cation channel in pig aortic endothelial cells. It is not clear, however, that this channel plays an important role in shear stress-stimulated alterations in endothelial cell metabolism because the force required to open this channel is much larger than the physiological range of shear stresses. Additional e v i d e n ~ eindicates ~ ~ . ~ ~ that shear stress activates a potassium channel that causes membrane hyperpolarization (FIGURE1).

FOS, JUN, CREB, AND THE LEUCINE ZIPPER

Cells can respond to external stimuli by the selective induction of immediate early genes (IEGs). The IEGs are characterized by extremely low expression in unstimulated cells; however, mRNA from these genes is detectable within minutes after stimulation. The induction of these genes is transient and the mRNAs typically have very short half-lives. Also, the induction of these genes does not depend on new protein synthesis. The members of two proto-oncogene families, fos and jun, appear to be closely linked to signal transduction pathways in several cell types, including endothelial , ~ ~j ~ n - D . ~The ~ . ~fos ’ cells. The jun family has three members: c - j ~ n ,j*~~ n - B and family consists of four genes: c - ~ o ~ , * ~ f o s - B , ~ ~andf1-u-2.~~ f i u - l , 3 ~ With the exception of jun-D, which is constitutively expressed and may be involved in the transcription of “housekeeping” genes32or responsible for basal transcription of other genes,33 all members of thefos and jun families are IEGs. c-fos,fos-B, c-jun, and jun-B mRNAs are detectable within minutes of cellular stimulation, have half-lives on the order of 20 minutes, and usually return to basal levels within 2 hours of stimulation. Alternative splicing of the fos-B mRNA gives rise to an additional mRNA species (Afos-B) that has similar kinetics to those of ~ O S - B Detectable . ~ ~ levels of fiu-1 and fru-2 mRNAs are not present until about 30 to 60 minutes after cellular stimulation and they remain elevated for as long as 4 hours before decreasing ~ i g n i f i c a n t l y . ~ ~ ~ ~ ~ Specific agents or signaling pathways that lead to expression of fos and jun family genes are summarized in TABLE1. Individual gene products of the fos and jun families form protein dimers (Fos/Jun complex) that bind to TRE (Tumor promoting agent Response Element) sites and to related sequences such as the CRE (CAMP Response Element) site on gene promoter DNA. The consensus sequence for the TRE is TGACTCA; the consensus sequence for the CRE is TGACGTCA. Any jun family protein (c-Jun, Jun-B, Jun-D) can dimerize with any gene product of thefos and jun families. However, fos family proteins (c-Fos, Fos-B, AFos-B, Fra-1, Fra-2) can only dimerize with members of the jun family. Dimerization of Fos and Jun proteins takes place through a structural motif that

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TABLE1. Expression of fos and jun Genes Gene c-jun jun-B jun-D c-fos fos-B fra-1 fra-2

cAMP/PKA yesh8 weakh9 weak69 ye~6~

Growth Factors/Serum yes70 yes69 weakz7 yes69 yes29 yes69 yes31

Phorbol Esters/PKC yes70 yesa weakz7 yesb9 weak29 yesb9

Membrane Depolarization noM) yesm yes60

has been termed the “leucine zipper”.36 These proteins and others contain a sequence of 29 amino acid residues that form an amphipathic a-helix (3.6 amino acids/turn of helix). Typically, every seventh amino acid in this sequence is a leucine residue. The leucine residues align along one side of the helix on every other loop, creating a hydrophobic face. This a-helix is referred to as the “zipper region” of Fos and Jun. The original model for the leucine zipper envisioned two antiparallel a-helices in which the leucine residues were interdigitated (hence, a zipper).36More recently, it has been determined that the two a-helices align in parallel, forming a coiled coil structure in which corresponding leucines from each monomer interact, but do not i ~ ~ t e r d i g i t a t eAn . ~ ~amino . ~ ~ acid sequence of about 30 mostly basic amino acids is located immediately adjacent to the amino-terminal end of the zipper region (see FIGURE2). Mutational studies have demonstrated that the zipper region facilitates dimerization, that the basic domain contacts the DNA, and that dimerization must take place before DNA binding can occur.394 Additionally, recent data indicate that the nonleucine residues in the zipper region determine the specificity of d i m e r i ~ a t i o nand ~ ~ may also account for the differing stabilities of Fos/Jun dimers. c-Fos/c-Jun heterodimers exhibit greater DNA binding activity because they are more stable than c-Jun/c-Jun homodimers.4w8 The combination of selective induction, differential kinetics, varying dimer stability, posttranslational modifications of Fos and J u ~ ,and ~ ~the, potential ~ ~ for preferential binding of individual Fos/Jun complexes to specific TRE and TRE-like sequences leads to numerous potential control mechanisms for gene expression. The CAMP Response Element Binding Protein (CREB) is a dimer that binds to the CRE. CREB can bind to the c-jun promoter blocking its expression even in the presence of serum and phorbol esters. Phosphorylation of CREB by a CAMPdependent protein kinase reverses this repression, acting as a molecular The carboxy-terminal end of the CREB monomer contains both a basic domain and a leucine zipper region that are homologous to the corresponding regions of the c J u n protein.51As with c-Jun, dimerization of CREB monomers takes place through the leucine zipper motif and the basic domain recognizes the target DNA (i.e., the CRE site).52Despite these similarities in the leucine zipper and basic domains of Fos/Jun and CREB and the homology shared by their target DNA sites, CREB does not bind to TRE sites even though Fos/Jun can bind to CRE site^.^^,^^ Synthetic peptides comprising the basic domains of c-Fos/c-Jun have a fourfold higher affinity for the CRE than synthetic peptides of the analogous region of CREB,5Ssuggesting

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that the Fos/Jun complex may be able to displace CREB under the appropriate cellular conditions.

PROTEIN SYNTHESIS IN MECHANICALLY STRESSED ENDOTHELIAL CELLS The alterations in protein synthesis and genetic expression that occur in mechanically stimulated endothelial cells may be mediated by members of the fos and jun families. Evidence for this hypothesis already exists. It has been demonstrated that expression of cfos can be induced in cardiac myocytes that are subjected to passive ~ t r e t c h i n g Whether .~~ other proto-oncogenes are expressed o r whether expression occurs in other cell types exposed to mechanical stimulation has not been established. By comparing the known changes in protein synthesis in shear stressstimulated endothelial cells with the known stimulation of second messenger systems in these cells, we can develop a hypothesis for the regulation of the altered genetic expression.

C-FOS

-

c-Jun

-

5' T G A C T C A 3' 3 ' - A C T G A G T - 5' TRE site on

target DNA

FIGURE 2. A schematic illustration of the interaction between the Fos and Jun proteins and the target DNA. The individual proteins first associate through the leucine zipper motif, which acts to stabilize the complex so that the basic domains can recognize the T R E site on the target DNA. Note that the consensus sequence for the T R E site is a palindrome, with the complementary sequence being nearly identical to the sequence for the T R E site.

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Endothelial cells exposed to shear stress produce elevated levels of tPA, whereas PAI-1 production remains ~naltered.23~ This is similar to the response observed in cells simultaneously exposed to phorbol esters, which stimulate protein kinase C activity by mimicking diacylglycerol, and forskolin, which is a CAMP a g o n i ~ tThis .~~ suggests that both the protein kinase C and the CAMPsignal transduction pathways may play a role in shear stress signal transduction. The promoter regions in the genes for tPA and PAI-1 (as well as ET, vWF, and TGF-P1) have sequences with homology to the CRE and TRE binding sites (see FIGURE3). Activation of either CAMPdependent protein kinases or protein kinase C can lead to the expression of fos and jun family genes (see TABLE 1). Endothelial cells exposed to another type of mechanical stress, namely, that induced by cyclical axial strain, respond by increasing production of prostacyclin and ET, whereas tPA production remains In vitro studies have demonstrated that cyclical stretching of endothelial cells results in depolarization of the cell membrane (as opposed to membrane hyperpolarization in endothelial cells exposed to shear Membrane depolarization has been shown to induce expression of c-fos and jun-B in PC12 cells without coinduction of c - j ~ n It . ~is also known that c-Fos/Jun-B dimers can suppress the transcription of genes whose promoters have only a single TRE site, whereas tram-activating genes whose promoters contain multiple TRE sites.61 Interestingly, the human tPA promoter contains a single TRE/CRE-like site and the human ET promoter contains three TRE-like sites (see FIGURE3). Unfortunately, the data for protein synthesis in endothelial cells exposed to cyclical stretching were obtained with bovine aortic endothelial cells and the promoter sequences for bovine tPA and ET are not known. However, if we assume that the bovine tPA and ET promoters contain the same functional elements as their human counterparts and that membrane depolarization induces c-fos and jun-B expression without coinduction of c-jun in depolarized endothelial cells as in depolarized PC12 cells, we can construct a simple signal transduction hypothesis as follows: (1) cyclic stretching of the endothelial cells changes membrane potential (i.e., depolarization); (2) as a consequence of membrane depolarization, c-fos and jun-B expression, but not c-jun expression, take place; (3) c-Fos/Jun-B dimers bind to the single TRE/CRE-like site in the tPA promoter, repressing transcription; (4) c-Fos/Jun-B dimers bind to all three TRE-like sites within the ET promoter, stimulating transcription. Thus, at the protein synthesis and secretion levels, the selective induction of IEGs can explain the results in endothelial cells exposed to cyclical stretching. An examination of the ET and PAL1 promoter sequences indicates that each contains three TRE-like sequences that are located at approximately the same positions with respect to their respective transcriptional start sites. If our signal transduction hypothesis is correct, then changes in PAI-1 production caused by mechanical stretching should be similar to those seen in E T production. Recent data generated in our laboratory suggest that PAI-1 secretion is increased in cyclically stretched endothelial cells,62consistent with this hypothesis. As noted, protein production in endothelial cells exposed to shear stress is quite different from protein production in cells exposed to cyclical stretching. Whereas ET and PAI-1 production are increased and tPA production is unchanged in stretched cells, tPA production is increased, endothelin production is decreased, and PAI-1

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Transcription start sites

7

+1

c-jun

P I TGACATCA

(-71I-64)

tPA [71]

TGACATCA

(-1 14/-105)

+1

ET

1721 TGCCTAA

TGGCTCA

TGACTAA

(-6561.650)

(-366/-360)

(-108/-102)

I +l

PAI-1 1731

5 TGACACA

TGAACACT

(-717/-711)

(-35U-349) (-15%-146)

TGCCTCA

TGACTCT (-41 8/-412)

TGAGACT

(+256/+262) +l

TGF- P I v41

I/\ TGTCTCA

TGAGACG

(-3711-365)

(+160/+167)

TGTCTCA

TGGCTCA

(-81 21-806)

(-35Z-346)

TGCCTCA

AGACTCA

(-826/-820)

(-55Z-546)

TGACTGA

(+302/+308)

FIGURE 3. The relative positions and sequences of potential TRE- and CRE-like sites in the genes for several endothelial cell proteins of interest. Transcriptional start sites are indicated by the vertical bars on the right-hand side and are numbered +1 for all genes. The consensus sequence for the TRE site is TGACTCA; for the CRE site, it is TGACGTCA. Note that the numbers in the brackets following the gene name denote the reference for the complete promoter sequence.

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production is unaffected in sheared endothelial cells. Thus, the response of endothelial cells to cyclic stretching is essentially opposite to that of endothelial cells exposed to shear stress. One potential mechanism to explain the data in endothelial cells exposed to shear stress is that c-fos and jun-B expression are induced without coinduction of c-jun as hypothesized in stretched endothelial cells, but where an additional factor interacts with, or blocks the interaction between, c-Fos/Jun-B heterodimers. An example for this type of mechanism involves the glucocorticoid receptor. Interaction between the glucocorticoid hormone-receptor complex and c-Fos can block c-Fos-mediated trans-activation of TRE-dependent t r a n ~ c r i p t i o n . ~ ~ Other examples of interactions between c-Fos and glucocorticoid hormone-receptor complex modulation of genetic expression exid3sa and a similar interaction takes place between c-Fos and the retinoic acid receptor.65 Additionally, if our signal transduction model for the stretched endothelial cells is correct, then a high glucocorticoid concentration in the extracellular environment should reverse the reported results (i.e., ET production should be down-regulated). Transcription factors other than the c-Fos/c-Jun heterodimer may be involved in the mechanical stress modulation of genetic expression in endothelial cells. In fact, the promoter regions for some of the members of the fos and jun families contain binding sites for other proto-oncogenes (e.g., jun-B contains a potential zif 268 binding site25.66),whereas other proto-oncogenes may contain binding sites for the Fos/Jun complex (c-jun has a bona fide TRE site6’). This raises the very intriguing question of how the regulators themselves are regulated.

SUMMARY There is increasing evidence that endothelial cells respond to the initiation of mechanical stress by the generation of certain second messengers and the activation of specific metabolic pathways. These rapid alterations in cellular function are accompanied by alterations in protein synthesis that are detectable several hours after initiation of the mechanical stress. The molecular mechanisms by which changes in the cytosol are converted to altered genetic expression in the nucleus are not known. Because agonist-induced modulations in the rate of synthesis of tPA and ET have been associated with the Fos and Jun protein families, it seems reasonable to propose that genetic expression in shear stress- or mechanical strain-stimulated endothelial cells is also regulated by selective induction offos and jun gene products. Testing of this hypothesis is actively under way in our laboratory. REFERENCES 1. F U R C H GR. O F. ~ ,& P. M. VANHOUTTE. 1989. FASEB J. 3: 2007-2018. 2. DIAMOND, S. L., S. G. ESKIN& L. V. MCINTIRE. 1989. Science 243: 1483-1485. S. L., J. B. SHAREFKIN, C. DIEFFENBACH, K. FRASIER-SCOTT, L. V. MCINTIRE & 3. DIAMOND, S. G. ESKIN.1990. J. Cell. Physiol. 143: 364-371. 4. SHAREFKIN, J. B., S. L. DIAMOND, S. G. ESKIN,L. V. MCINTIRE& C. W. DIEFFENBACH. 1991. J. Vasc. Surg. 14: 1-9. & G. JOHNSON. 1988. J. Vasc. Surg. 7: 130-138. 5. SUMPIO, B. E., A. J. BANES,M. BUCKLEY 6. HSIEH,H., N. Q. LI & J. A. FRANGOS. 1991. Am.J. Physiol. 260 H642-H646.

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Regulation of genetic expression in shear stress-stimulated endothelial cells.

There is increasing evidence that endothelial cells respond to the initiation of mechanical stress by the generation of certain second messengers and ...
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