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Molecular Endocrinology, doi:10.1210/me.2004-0279
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Molecular Endocrinology 19 (4): 1012-1023
Copyright © 2005 by The Endocrine Society

Relaxin Stimulates Protein Kinase C {zeta} Translocation: Requirement for Cyclic Adenosine 3',5'-Monophosphate Production

Bao T. Nguyen and Carmen W. Dessauer

Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030

Address all correspondence and requests for reprints to: Carmen W. Dessauer, Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, Texas 77030. E-mail: Carmen.W.Dessauer{at}uth.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Relaxin is a polypeptide hormone that activates the leucine-rich repeat containing G protein-coupled receptors, LGR7 and LGR8. In an earlier study, we reported that relaxin produces a biphasic time course and the second wave of cAMP is highly sensitive to phosphoinositide-3 kinase inhibitors (LY294002 and wortmannin). LY294002 inhibits relaxin-mediated increases in cAMP production by 40–50% across a large range of relaxin concentrations. Here we show that protein kinase C {zeta} (PKC{zeta}) is a component of relaxin signaling in THP-1 cells. Sphingomyelinase increases cAMP production due to the release of ceramide, a direct activator of PKC{zeta}. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin induced cAMP production to the same degree (~40%) as LY294002. Relaxin stimulates PKC{zeta} translocation to the plasma membrane in THP-1, MCF-7, pregnant human myometrial 1–31, and mouse mesangial cells, as shown by immunocytochemistry. PKC{zeta} translocation is phosphoinositide-3 kinase dependent and independent of cAMP production. Antisense PKC{zeta} oligodeoxynucleotides (PKC{zeta}-ODNs) deplete both PKC{zeta} transcript and protein levels in THP-1 cells. PKC{zeta}-ODNs abolish relaxin-mediated PKC{zeta} translocation and inhibit relaxin stimulation of cAMP by 40%, as compared with mock and random ODN controls. Treatment with LY294002 in the presence of PKC{zeta}-ODNs results in little further inhibition. In summary, we present a novel role for PKC{zeta} in relaxin-mediated stimulation of cAMP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TRADITIONALLY KNOWN AS a hormone of pregnancy, the polypeptide hormone relaxin has pleiotropic effects on reproductive tissues during pregnancy including growth and softening of the cervix, inhibition of uterine contractility, growth and development of the mammary apparatus, vasodilation of blood vessels, and regulation of cardiovascular function (1). Relaxin-deficient mice showed inadequate development in the reproductive tract (2) and progressive tissue fibrosis (3). The latter phenotype and the matrix remodeling activity of relaxin have sparked interest in relaxin as a potential antifibrotic agent. In human dermal fibroblasts, relaxin decreased fibrosis (4) and in culture systems, relaxin increased turnover of collagen (5) and fibronectin (6). In rodent models of fibrosis, relaxin decreased collagen accumulation and other markers of fibrosis in lung, kidney, liver, and heart (7, 8, 9, 10, 11).

Despite the long history of relaxin, the molecular mechanism(s) of relaxin signaling have been elusive. This changed dramatically with the identification of the relaxin receptors as the leucine-rich repeat containing G protein-coupled receptors, LGR7 and LGR8 (12). Early studies described increased cAMP and protein kinase A activity upon relaxin treatment (13, 14, 15, 16, 17, 18). This was confirmed with the overexpression of LGR7 and LGR8, leading to a large increase in cAMP upon addition of relaxin (12). Additional signaling pathways are activated by relaxin as well. Relaxin activated MAPK in human endometrial and THP-1 monocytic cells (19) and stimulated nitric oxide production in human vascular smooth muscle and breast cancer cells (20, 21). Relaxin also stimulated phosphoinositide-3 kinase (PI3K), which enhanced cAMP production by adenylyl cyclase (AC) in THP-1 cells (22). It is currently unclear, which signaling pathway(s) contributes to the many relaxin phenotypes.

To further understand the enhancement of relaxin-stimulated cAMP production by PI3K, we searched for downstream targets of PI3K that could activate AC. A number of known downstream targets of PI3K have been reported including AKT/protein kinase B, Btk (Bruton tyrosine kinase), PDK (phosphoinositide-dependent kinase), Vav/Rac (guanine nucleotide exchange factor/small G protein), and PKC{zeta} (protein kinase C {zeta}) (23, 24). Among these targets, PKC{zeta} was a prime candidate because it activates type II and V AC (25, 26, 27). PKC{zeta} is an atypical isoform of the PKC superfamily in which PKCs are classified into three groups based on their allosteric activators. Conventional PKCs ({alpha}, ßI, ßII, and {gamma}) are activated by diacyglycerol and Ca2+ and novel PKCs ({delta}, {epsilon}, {eta}, µ, and {theta}) by diacyglycerol (28, 29, 30). The atypical PKCs ({iota}/{lambda} and {zeta}) are directly or indirectly activated by phosphatidylinositol 3,4,5-triphosphate (PIP3) and other lipids, but not by diacylglycerol and Ca2+ (31, 32, 33).

In the current study, we present a novel role for PKC{zeta} in the relaxin-mediated stimulation of cAMP. Our results show that relaxin stimulates PKC{zeta} translocation to the plasma membrane in a number of cell lines, known to respond to relaxin treatment including human breast cancer [MCF-7 (34)], pregnant human myometrial [PHM1-31 (35, 36)], mouse mesangial [MMC (6)], and human monocytic [THP-1 (18, 37, 38)] cell lines. Relaxin-stimulated PKC{zeta} translocation is dependent on PI3K and independent of cAMP. Finally, PKC{zeta} is required for the enhancement of relaxin-stimulated cAMP production by PI3K.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Relaxin Dose-Response Curve in THP-1 Cells
In an earlier study using THP-1 cells, we reported that porcine relaxin (0.5 µg/ml) produced a biphasic time course of cAMP in which the first peak appeared as early as 1–2 min with a second wave of cAMP generated at 10–20 min (22). The second wave of cAMP was highly sensitive to inhibitors of PI3K (LY294002 and wortmannin). We have now examined the components, downstream of PI3K, required for increasing cAMP in response to relaxin. A 20-min treatment with porcine relaxin stimulated cAMP production across a large range of relaxin concentrations from 0.1–100 ng/ml (EC50 ~3.4 ng/ml) (Fig. 1AGo, top panel). LY294002 inhibited the stimulation of cAMP production by 40–50% from 1 ng/ml to 100 ng/ml of relaxin (Fig. 1AGo, bottom panel). Concentrations below 1 ng/ml were likely not inhibited to the same degree by LY294002 due to the large contribution of basal cAMP levels. Therefore, relaxin couples to both AC and PI3K at physiological concentrations of relaxin, which range from 3.7 to over 800 ng/ml in plasma of pregnant hamster (39) and peak at 0.8–1.2 ng/ml in serum of women during pregnancy (1).



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  		Fig. 1. Role of PI3K and PKC in Relaxin-Mediated Increases in cAMP in THP-1 Cells

A (Top panel), THP-1 cells (duplicate samples of 3 x 105 cells per treatment) were pretreated in the presence of PDE inhibitor (50 µM IBMX) with or without PI3K inhibitor (50 µM LY294002; LY) for 15 min, and then treated with vehicle or porcine relaxin (0.1 to 100 ng/ml) for 20 min. A (Bottom panel), Percent inhibition by LY. B, THP-1 cells (duplicate samples of 3 x 105 cells per treatment) were pretreated with 50 µM IBMX for 15 min, and then treated with vehicle, 100 mU/ml sphingomyelinase (SGM), or relaxin (100 ng/ml) for 20 min. Significant differences (*, P < 0.05; **, P < 0.001) compared with vehicle treatment are designated (t test). C, THP-1 cells (duplicate samples of 3 x 105 cells per treatment) were pretreated with 50 µM IBMX in the presence or absence of chelerythrine chloride (0.2 to 5 µM) for 15 min, and then treated with vehicle, relaxin (100 ng/ml), or forskolin (10 µM) for 20 min. Reactions were stopped upon addition of HCl (0.1 N final) and total cAMP accumulation was measured by enzyme immunoassay as described in Materials and Methods. Data are expressed as the mean ± SE from a single experiment and are representative of four (A) and two (B and C) separate experiments.

 
An Isoform of PKC Enhances Relaxin Stimulation of cAMP in THP-1 Cells
Although we know that PI3K is required for the full stimulation of cAMP by relaxin (22), the mechanism of PI3K-mediated activation of AC is unknown. Among the many downstream targets of PI3K, PKC{zeta} was a likely candidate because it activates type V AC both in vitro and upon overexpression in vivo (25, 26). PKC{zeta} is an atypical isoform of PKC. Although regulation of PKC{zeta} is not completely understood, it is insensitive to diacylglycerol and Ca2+ but is activated by ceramide, a lipid product generated from sphingomyeline by sphingomyelinase. Relaxin increases PI3K activity, and thus the production of PIP3 (22), which directly or indirectly stimulates PKC{zeta} activity (23).

Currently, there are no selective inhibitors available for PKC{zeta}. As a result, we first used a number of nonselective pharmacological agents to determine whether any isoform of PKC is downstream of PI3K for relaxin stimulation of cAMP in THP-1 cells. Sphingomyelinase treatment (100 mU/ml) results in more than a 2-fold increase in cAMP production (Fig. 1BGo). The ceramide generated from sphingomyelinase treatment would not be expected to increase cAMP to the same extent as relaxin. First, the release of ceramide is slow compared with relaxin stimulation of cAMP. Second, and more importantly, relaxin utilizes both G{alpha}s and PI3K to enhance cAMP production. If this dual activation is mediated through PKC{zeta}, it should be highly synergistic as shown previously for PKC{zeta} activation of ACV. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin (100 ng/ml) in a dose-dependent manner. There was little effect on basal cAMP levels, and chelerythrine chloride either had little effect or even increased forskolin-stimulated cAMP production (Fig. 1CGo), ruling out general actions on direct AC activity. Therefore, these results suggest that the PKC{zeta}-AC pathway is functional and an isoform of PKC may be required for full relaxin stimulation in THP-1 cells.

Relaxin Stimulates PKC{zeta}/{iota} Translocation in THP-1, MCF-7, PHM1–31, and MMC Cells
PKC{zeta} is found in the cytosol in the inactive state. Upon stimulation, PKC{zeta} is translocated to the plasma membrane by the generation of PIP3 (32, 33, 40, 41). In human monocytes, lipopolysaccharide (LPS) activated PKC{zeta} via PI3K (42) and induced adherence in THP-1 cells in a PI3K-dependent manner (43). Thus, LPS treatment was used as a positive control to activate PKC{zeta} in THP-1 cells (Fig. 2AGo). Unfortunately, all commercially available polyclonal antibodies for PKC{zeta} also recognize the atypical PKC{iota} as well. Thus, immunofluorescence labels both PKC{zeta} and PKC{iota}, designated as PKC{zeta}/{iota}. This issue is resolved in later experiments shown in Figs. 5Go and 6Go. Fluorescence microscopy showed that relaxin (100 ng/ml) stimulated PKC{zeta}/{iota} translocation to the plasma membrane in THP-1, MCF-7, PHM1–31, and MMC cells (Figs. 2–4GoGoGo, Table 1Go, and data not shown). This result is consistent with a previous report of an increase in PKC activity in the membrane fraction of relaxin-stimulated human endometrial cells (44). Translocation was dose dependent and, in all cell lines tested, relaxin-stimulated translocation was blocked by preincubation with the PI3K inhibitor, LY294002 (Figs. 2AGo, 3AGo, and 4BGo). The time course for translocation suggested this occurred as early as 2 min for a duration of more than 10 min in PHM1–31, and THP-1 cells (Fig. 4AGo and data not shown).



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  		Fig. 2. Relaxin Stimulates PKC{zeta}/{iota} Translocation to the Plasma Membrane in THP-1 Cells

A, THP-1 cells were pretreated with or without PI3K inhibitor (50 µM LY294002; LY) for 15 min, and then treated with vehicle, relaxin (100 ng/ml), or LPS (1 µg/ml) for 5 min. Cells were fixed and stained with anti-PKC{zeta} antibody as described in Material and Methods. Representative overlays of DAPI- and Cy2-stained sections from the same experiment are shown and similar results were observed in five different experiments. B, THP-1 cells were treated with vehicle or relaxin (100 ng/ml) for 3 min. Representative confocal images of THP-1 cells from the same experiment stained for PKC{zeta}, Na+/K+ ATPase, or an overlay between PKC{zeta} and Na+/K+ ATPase, are shown for vehicle and relaxin treatment and are representative of multiple images from two separate experiments. C, THP-1 cells were washed with prewarmed PBS and starved in 1% FBS/RPMI 1640 containing 2 mM L-glutamine for 36–48 h. Duplicated samples were treated with vehicle, relaxin (500 ng/ml), LPS (0.5 µg/ml) for 10 min. Treatments were stopped with ice-cold PBS, and cells were lysed, fractionated, and subjected to Western blotting with polyclonal rabbit anti-PKC{zeta} (C20) antibody as described in Materials and Methods. Upper graph represents data from three separate experiments performed in duplicate and are expressed as the mean ± SE. Significant differences shown between groups using one-way ANOVA analysis (P < 0.05, n = 3) are designated by different lowercase letters. A representative blot of the membrane fraction from a single experiment is also shown.

 


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  		Fig. 5. Antisense PKC{zeta} Oligodeoxynucleotides Deplete PKC{zeta} Transcript and Protein Levels in THP-1 Cells

THP-1 cells (1.0 x 106 cells per treatment) were transfected with mock, random oligodeoxynucleotide (Ran-ODNs; 1 µM), or antisense PKC{zeta} oligodeoxynucleotides (PKC{zeta}-ODNs; 0.125–1 µM) for 48 h. A, Cells were harvested and PKC{zeta} transcript levels were detected using RT-PCR. B, PKC{zeta}/{iota} protein levels were detected using SDS-PAGE and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. Data from each experiment are expressed as the mean ± SE from a single experiment and are representative of at least four different experiments for each set. Significant differences (*, P < 0.05) between groups are designated (t test).

 


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  		Fig. 6. Antisense PKC{zeta} Oligodeoxynucleotides Abolish Relaxin-Mediated PKC{zeta} Translocation in MCF-7 Cells

MCF-7 cells (4.0 x 104 cells per well) were transfected with mock, 2 µM random oligodeoxynucleotides (Ran-ODNs), or 0.25–2 µM antisense PKC{zeta} oligodeoxynucleotides (PKC{zeta}-ODNs) for 48 h. Each set of cells were treated with 100 ng/ml relaxin for 5 min, fixed, and stained as described in Materials and Methods. Representative overlays of DAPI- and Cy2-stained sections from the same experiment are shown and similar results were observed in two different experiments.

 


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  		Fig. 3. Relaxin Stimulates PKC{zeta}/{iota} Translocation to the Plasma Membrane in MCF-7 Cells

A, MCF-7 cells were pretreated with or without PI3K inhibitor (50 µM LY294002; LY) for 15 min, and then treated with vehicle or relaxin (100 ng/ml) for 5 min. B, MCF-7 cells were treated with vehicle, relaxin (100 ng/ml), or forskolin (10 µM) for 5 min. Cells were fixed and stained with anti-PKC{zeta} antibody as described in Material and Methods. Representative overlays of DAPI- and Cy2-stained sections from the same experiment are shown and similar results were observed in five (A) and two (B) different experiments. C, Representative confocal images (nos. 2–4 of eight total images per stack; 0.4 µm each) of MCF-7 cells stained with anti-PKC{zeta} antibody are shown for vehicle and relaxin treatment (100 ng/ml; 5 min) from the same experiment and are representative of multiple cells from two different experiments.

 


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  		Fig. 4. Relaxin Stimulates PKC{zeta}/{iota} Translocation in PHM1–31 and MMC Cells

A, PHM1–31 cells (4 x 104 cells per well) were treated with vehicle or 100 ng/ml relaxin for the indicated times. B, MMC cells (4 x 104 cells per well) were pretreated with dimethylsulfoxide (DMSO) or 50 µM LY294002 for 15 min followed by vehicle or 100 ng/ml relaxin for 2 or 5 min. Cells were fixed and stained as described in Materials and Methods. Representative overlays of DAPI- and fluorescein isothiocyanate (PHM1–31) or Cy2 (MMC)-stained sections from the same experiment are shown and similar results were observed in two different experiments.

 

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  		Table 1. Relaxin Stimulation of cAMP and PKC{zeta}

 
Translocation of PKC{zeta}/{iota} to the plasma membrane was confirmed using confocal microscopy in THP-1 and MCF-7 cells (Figs. 2BGo and 3CGo). Using the Na+/K+ ATPase as a plasma membrane marker, we were able to semiquantitate the localization of PKC{zeta}/{iota} at the plasma membrane upon relaxin treatment in THP-1 cells (Fig. 2BGo). Under basal conditions, 10.5% of the PKC{zeta}/{iota} immunofluorescence was colocalized with the Na+/K+ ATPase. Relaxin treatment increased PKC{zeta}/{iota} colocalization with the plasma membrane marker by almost 3-fold (29.3%).

Blinded analysis of THP-1 and MCF-7 translocation experiments revealed that a significant number of cells exhibited a PKC{zeta}/{iota} plasma membrane phenotype in response to relaxin (THP-1: basal 15/74 cells (20%) vs. relaxin 66/89 cells (74%), n = 3; MCF-7: basal 23/121 cells (19%) vs. relaxin 218/314 cells (69%), n = 5). In addition, subcellular fractionation of THP-1 cells demonstrated a 1.7-fold increase in PKC{zeta}/{iota} protein present in the membrane fraction after whole cell treatment with relaxin (Fig. 2CGo).

Although most of the cell lines tested produced at least small increases in cAMP upon relaxin treatment (Table 1Go), PKC{zeta}/{iota} translocation was not mediated by cAMP. Neither isoproterenol nor forskolin treatment resulted in PKC{zeta}/{iota} translocation in MCF-7 cells, although these treatments produced an 8-fold increase in cAMP production (Fig. 3BGo and data not shown). Therefore, relaxin-stimulated PKC{zeta}/{iota} translocation is dependent on PI3K and independent of cAMP production.

PKC{zeta} Antisense Oligodeoxynucleotides Deplete PKC{zeta} Transcript and Protein Levels in THP-1 Cells
To assess the involvement of PKC{zeta} in the relaxin signaling pathway, we used antisense PKC{zeta} phosphorothioate oligodeoxynucleotides (PKC{zeta}-ODNs) targeting the internal sequence of PKC{zeta} transcript (45) to deplete PKC{zeta} in THP-1 cells. THP-1 cells are notoriously difficult to transfect with plasmids, limiting the usefulness of dominant-negative constructs. After 48 h of treatment, PKC{zeta}-ODNs selectively deplete PKC{zeta} transcript, but not PKC{iota} transcript, another atypical isoform of PKC (Fig. 5AGo). Despite the fact that our polyclonal antibodies recognize both PKC{zeta} and PKC{iota}, approximately 40% of PKC{zeta}/{iota} immunoreactivity is depleted by PKC{zeta}-ODNs treatment (Fig. 5BGo). Thus, PKC{zeta}-ODNs treatment depleted both PKC{zeta} transcript and protein levels in THP-1 cells.

PKC{zeta}-ODNs Abolish Relaxin-Stimulated PKC{zeta} Translocation in MCF-7 Cells and Inhibits Relaxin-Mediated cAMP Production in THP-1 Cells
The depletion of PKC{zeta} transcript and protein levels by PKC{zeta}-ODNs now provided a specific pharmacological agent to examine the role of PKC{zeta} in relaxin signaling. As an additional control, we determined whether the PKC{zeta}-ODNs could block the observed translocation of PKC{zeta}/{iota} to the plasma membrane. PKC{zeta}-ODNs abolished relaxin-stimulated PKC{zeta} translocation observed in MCF-7 cells in a dose-dependent manner and significantly decreased the overall levels of immunofluorescence (Fig. 6Go). Therefore, the immunofluorescence signal giving rise to relaxin-mediated translocation was not due to any cross-reactivity with PKC{iota} and was specific for PKC{zeta}. In addition, PKC{zeta}-ODNs inhibited relaxin-mediated cAMP production by 40% compared with treatment with random-ODNs or mock conditions in THP-1 cells (Fig. 7Go). Pretreatment with LY294002 in THP-1 cells transfected with PKC{zeta}-ODNs resulted in little further inhibition of relaxin-stimulated cAMP production (Fig. 7Go). Therefore, PKC{zeta} is clearly necessary for full stimulation of cAMP by relaxin, providing an important link between PI3K and AC activation.



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  		Fig. 7. Antisense PKC{zeta} Oligodeoxynucleotides Inhibit Relaxin-Stimulated cAMP Production in THP-1 Cells

THP-1 cells (1 x 106 cells per well) were transfected with mock, 1 µM random oligodeoxynucleotide (Ran-ODNs), or 0.125–1 µM antisense PKC{zeta} oligodeoxynucleotides (PKC{zeta}-ODNs) at the indicated concentrations for 48 h. Cells (duplicate samples of 2 x 105 cells per treatment) were pretreated with 50 µM IBMX in the presence or absence of PI3K inhibitor (50 µM LY294002; LY) for 15 min. Cells were then treated with vehicle or 100 ng/ml relaxin for 20 min before reactions were stopped and analyzed for cAMP accumulation. Data are expressed as the mean ± SE from a single experiment and are representative of at least four different experiments. Significant differences (*, P < 0.05) within a group are designated (t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have shown previously that relaxin stimulates PI3K activity, which in turn enhances adenylyl cyclase activity producing a second wave of cAMP production in THP-1 cells. We proposed a bifurcated model whereby relaxin binding to its receptor(s) LGR7/8 activates Gs, leading to the activation of AC by two distinct mechanisms. The initial mechanism is a traditional G{alpha}s-membrane delimited activation of AC, resulting in rapid increases in cAMP. The second mechanism requires PI3K activation and is somewhat delayed as compared with G{alpha}s activation. Inhibitors of PI3K partially block relaxin-stimulated cAMP production across a wide range of physiological relaxin concentrations (1–500 ng/ml).

The question still remained as to how does PI3K increase the accumulation of cAMP? One attractive possibility was that PI3K activated atypical PKC{zeta} (either directly or indirectly), which in turn phosphorylated AC to stimulate activity. PKC{zeta} is a well-known downstream effector of PI3K and has been previously shown to stimulate AC activity. Type V AC activity is stimulated 20-fold in the presence of purified PKC{zeta} in vitro or by transfection of PKC{zeta} in whole cells (25, 26). Activation of type II AC in whole cells by lysophosphatidic acid may also be mediated by atypical PKC isoforms (27). It had previously been shown that relaxin treatment increased the proportion of PKC activity found in the membrane fraction of human endometrial cells (44), thus stimulating our interest in PKC as a downstream signaling molecule.

The generation of PIP3 by PI3K results in the translocation of PKC{zeta} to the plasma membrane. There are two proposed mechanisms for translocation. PKC{zeta} may be directly recruited to the plasma membrane by PIP3 via the PH domain on PKC{zeta} or indirectly recruited to the plasma membrane by formation of a complex with PDK (32, 33, 40, 41). Relaxin stimulated a clear redistribution of PKC{zeta} from the cytosol to the plasma membrane in THP-1, MCF-7, PHM1–31, and MMC cells. All of these cell lines are known to respond to relaxin. Relaxin increased cAMP and vascular endothelial growth factor (VEGF) mRNA in THP-1 cells (38, 46); differentiated MCF-7 cells (47); inhibited oxytocin-stimulated PI turnover and rise in Ca2+, and activated maxi-K channels in PHM1–31 cells (48); and degraded fibronectin and collagen in MMC cells (6). Although relaxin stimulated translocation of PKC{zeta} and the differentiation in MCF-7 cells (34), relaxin did not stimulate detectable increases in cAMP in these cells, even in the presence of low forskolin concentrations to enhance synergy. Despite a lack of cAMP response in MCF-7, LGR7, and/or LGR8 mRNA are detected in all of these cell lines by RT-PCR (Ref.22 and data not shown). These cells may possess high basal cAMP and generate only local cAMP signals, or have high levels of phosphodiesterases insensitive to the general phosphodiesterase (PDE) inhibitors used. Alternatively, relaxin may be preferentially coupled to PI3K-dependent signaling pathways in MCF-7 cells. Further studies are required to determine the relative signaling by relaxin to cAMP, MAPK, or PI3K signaling pathways in different cell types.

PKC{zeta} is not only activated by relaxin, it is also required for the PI3K-dependent enhancement of cAMP production by relaxin. General PKC inhibitors such as chelerythrine chloride produced a partial inhibition of relaxin-stimulated cAMP production, similar to that observed with PI3K inhibitors (Fig. 1CGo). Sphingomyelinase treatment resulted in a 2-fold increase in cAMP, indicating that a PKC{zeta}-mediated pathway was at least present to increase cAMP production. Antisense PKC{zeta} oligonucleotides (PKC{zeta}-ODNs) were used to knock down PKC{zeta} mRNA and protein levels to demonstrate a role for PKC{zeta} in cAMP production by relaxin. PKC{zeta}-ODNs inhibited relaxin-stimulation of cAMP with no additional inhibition by PI3K inhibitors. Thus, PKC{zeta} is downstream of PI3K and contributes to the activation of adenylyl cyclase in THP-1 cells, which is likely synergistic with activation by G{alpha}s. This is the first example of PKC{zeta} regulation of AC in an endogenous system. The dual nature of the relaxin signaling pathway may account for many of the relaxin-mediated effects not easily explained by a simple Gs/G protein-coupled receptor signaling paradigm as discussed below.

Physiological Consequences of PKC{zeta} Activation
In addition to its potential effects on cAMP, PKC{zeta} is essential in smooth muscle cells for the activation of matrix metalloproteases (MMP)-1, -3, and -9 (45). In human uterine fibroblasts, relaxin increased MMP-1 and -3 expression (49), whereas MMP-9 expression was increased by relaxin in uterine, cervix and breast cancer cell lines (50, 51). The induction of MMPs is believed to be an important aspect of relaxin-induced matrix remodeling and antifibrotic actions (52, 53).

In conjunction with PI3K, PKC{zeta} is required for the transcriptional activation of VEGF in numerous systems including pancreatic and renal cancer cells, glioblastomas, retinal cells and keratinocytes (54, 55, 56, 57). Relaxin increased mRNA for VEGF in cells collected at wound sites, endometrial cells and in THP-1 cells (19, 46, 58). This was associated with the ability of relaxin to stimulate angiogenic effects at sites of ischemic wound healing and the neovascularization of the endometrial lining of the uterus (46, 59). Finally, mice deficient in PKC{zeta} have defects in ERK activation in lung, B cells, and other immune cells (60, 61). Relaxin stimulated ERK activation in THP-1 (a monocytic cell line) and pulmonary and coronary artery cells (19). The mechanism of relaxin-mediated activation of ERK or induction of MMPs or VEGF is currently unknown; however, it is tempting to speculate that the activation of PI3K and PKC{zeta} by relaxin may play an important role in modulating downstream events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Forskolin, 3-isobutyl-1-methylxanthine (IBMX), sphingomyelinase, and isoproterenol were obtained from Sigma (St. Louis, MO); LY294002 and chelerythrine chloride were obtained from Calbiochem (La Jolla, CA). Cell culture reagents and Lipofectamine 2000 were obtained from Invitrogen Life Technologies (Gaithersburg, MD). Enzyme immunoassay kit for cAMP was purchased from Assay Designs, Inc. (Ann Arbor, MI). Deoxyribonuclease (DNase), avian myeloblastosis virus (AMV) reverse transcriptase, and Taq DNA polymerase were obtained from Promega Corp. (Madison, WI).

Cell Culture
The human breast cancer cell line MCF-7 and the MMC cell line (62) were cultured at 37 C and 5% CO2 in DMEM/F-12 and DMEM, respectively. The medium was supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cultures of the immortalized pregnant human myometrial cell line PHM1–31 and the human monocytic cell line THP-1 have been described previously (22, 63).

Assay for cAMP Accumulation
Detailed procedures have been described previously (22). Briefly, cultured THP-1 cells were washed with prewarmed PBS, starved in media containing 1% FBS overnight, and resuspended with cell suspension media (RPMI 1640 + 50 mM HEPES, 7.4) at 37 C. Cells were pretreated with PDE inhibitor (50 µM IBMX) for 15 min, and then treated with 100 ng/ml relaxin for 20 min, except where indicated. Treatments were terminated by addition of 1 N HCl (0.1 N HCl final). Total cAMP (intra- and extracellular) was detected by enzyme immunoassay. Data represent mean ± SE and were analyzed by t test and one-way ANOVA analysis as indicated.

RT-PCR Analysis
Poly(A)+ RNA was isolated from human THP-1 cells, reverse transcribed, and amplified by PCR as previously described (12). Briefly, approximately 2 x 106 cells were harvested by centrifugation at 1500 rpm for 5 min, washed twice with cold PBS, and resuspended in RLT lysis buffer provided by RNeasy Mini Kit (QIAGEN, Valencia, CA). Poly(A)+ RNA was isolated with the RNeasy Mini Kit according to the manufacturer’s protocol and DNase-treated for 30 min at 37 C and 10 min at 75 C. cDNA synthesis was carried out in a final volume of 10 µl containing 0.5 µg of DNase-treated poly(A)+ RNA, 1x AMV buffer, 10 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphates, 4 µM reverse primer, nuclease-free water, and 0.45 U AMV reverse transcriptase. Reverse transcriptase conditions were 50 C for 30 min, 72 C for 10 min, and 4 C for 5 min. Touch-down PCR was carried out in a final volume of 50 µl containing 10 µl of cDNA, 1x PCR buffer, 1.5 mM MgCl2, 0.12 mM deoxynucleotide triphosphates, 0.05 µM reverse and forward primer, nuclease-free water, and 1.25 U Taq DNA polymerase. Touch-down PCR conditions were 94 C for 60 sec followed by 6 cycles (94 C for 15 sec, 66 C for 25 sec, and 72 C for 40sec), 27 cycles (94 C for 15 sec, 63 C for 25 sec, and 72 C for 40 sec), and final extension (72 C for 5 min). PCR products were electrophoresed on a 3.5% NuSieve/agarose (3:1) gel containing 0.5 µg/ml ethidium bromide and visualized under UV light. Primer sets used for amplification of PKC{zeta} and PKC{iota} were described previously (64).

Assay for PKC{zeta} Translocation: Immunocytochemical Staining Procedures
For adherent cell lines such as MCF-7, MMC, and PHM1–31, cells (4 x 104 cells per well) were seeded in a 24-well plate containing a cover glass (12 mm diameter) in each well approximately 24 h before use. For the suspension cell line THP-1, the following procedures were carried out to attach cells to the coverslip: THP-1 cells (1.0 x 106 cells per well) were washed once with prewarmed PBS, resuspended in RPMI 1640 containing 2 mM L-glutamine, seeded in six-well plate containing a cover glass (18 mm square) in each well, and serum-starved overnight. Before treatment, medium in each well was replaced with basal medium plus HEPES (pH 7.40 (final, 50 mM). Cells were treated with vehicle, 100 ng/ml relaxin, or 1 µg/ml LPS for 5 min or as indicated. Cells were then rinsed briefly with PBS, fixed in 100% methanol at –20 C for 10–20 min as indicated, and rinsed again in PBS three times, 10 min each. Procedures for immunocytochemical staining were as previously described for methanol-fixed cells (65) with the following modifications: methanol-fixed cells were incubated for 1 h at room temperature with the primary antibody against PKC{zeta} (5 µg/ml in PBS; polyclonal rabbit anti-PKC{zeta} C20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and/or Na+/K+ ATPase (10 µg/ml in PBS; mouse anti-Na+/K+ ATPase) (Upstate, Charlottesville, VA). Similar results were observed in cells preincubated with or without blocking solution (5% rabbit serum in PBS) before antibody incubation. Cells were washed three times, 10 min each in PBS before incubation with secondary antibody [Cy2-conjugated goat antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA); fluorescein isothiocyanate-conjugated goat antirabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); or Texas red-conjugated goat antimouse IgG for the Na+/K+ ATPase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA)]. After incubation with secondary antibody (2 µg/ml in PBS) for 45 min, cells were washed three times, 10 min each in PBS, blotted to remove as much PBS as possible, and mounted with mounting media containing 4',6-diamidino-2-phenylindole (DAPI) (Vector Shield, Burlingame, CA) for fluorescence microscopy or with 0.1 M N-propylgellate in 50% glycerol/PBS (66) for confocal microscopy on a superfrost slide (Fisher Scientific Co., Houston, TX). Sections were viewed and photographed using either an Olympus BX60 microscope equipped with a Hamamatsu digital camera (Leeds Instruments, Inc., Irving, TX) or an LSM 510 META (Carl Zeiss, Jena, Germany) for confocal images.

Digitized confocal images (x63 magnification) were processed for semiquantitative morphometric analysis using LSM 510 Meta version 3.2 SP2 software (Carl Zeiss). First, a level adjustment was performed to normalize intensities between channels, followed by analysis of a pixel histogram of selected cells. A mean value was calculated from several individual cells for the percent overlap of the total PKC{zeta}/{iota} intensity with the plasma membrane marker intensity of the Na+/K+ ATPase. In addition, immunocytochemical images were scored for PKC{zeta}/{iota} plasma membrane phenotypes of individual cells using blinded analysis by three different investigators, not affiliated with this project.

Assay for PKC{zeta} Translocation: Biochemical Fractionation
Cultured THP-1 were washed with prewarmed PBS, starved in 1% FBS for 36–48 h, and treated with vehicle or relaxin (500 ng/ml) for 10 min. Treatments (2 x 106 cells per treatment) were stopped with ice-cold PBS, and cells were lysed and fractionated as described previously (67). In brief, treated cells were resuspended in 300 µl of hypotonic fractionation buffer [10 mM Tris (pH 7.4), 4.5 mM EDTA, 2.5 mM EGTA, 2.3 mM 2-mercaptoethanol, 1.0 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 nM microcystin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin], incubated for 20 min at 4 C while rotating, and sonicated with three quick pulses [duty cycle 10 and output control 1.5 (Branson Sonifier 250)]. Total cell lysates were then centrifuged at 100,000 x g for 30 min at 4 C to separate cytosolic from particulate fractions. The resulting pellets were extracted in 150 µl of hypotonic fractionation buffer containing 0.5% Triton X-100 for 20 min and centrifuged at 16,000 x g for 20 min at 4 C to separate the detergent-insoluble and -soluble material. The resulting supernatant was taken to represent the membrane fraction. Equal amounts of total membrane protein for each sample (30–50 µg) was separated by SDS-PAGE and blotted with polyclonal rabbit anti-PKC{zeta} (C20) antibody.

Immunoblot Analysis
Cells were resuspended in cell lysis buffer [20 mM Tris (pH 7.5), 250 mM sucrose, 1.2 mM EGTA, 20 mM ß-mercaptoethanol, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM NaF, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotonin, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride] (68), lysed by passing through a syringe with a 23-gauge needle 10 times, and insoluble debris was removed by centrifugation. The supernatants (20–40 µg) were boiled for 10 min in Laemmli buffer, resolved by 8% SDS-PAGE, and transferred to nitrocellulose. Western blots were visualized by enhanced chemiluminescence.

Oligodeoxynucleotide Treatments
Antisense phosphorothioate PKC{zeta} oligodeoxynucleotides (PKC{zeta}-ODN) and random phosphorothioate oligodeoxy-nucleotides (Ran-ODN) were synthesized by Sigma Genosys (Woodlands, TX). Sequences of ODNs were AS-ODN: 5'-CGTCCTCGTTCTTG-3' and Ran-ODN: 5'-GCCTTATTTACTACTTTCGC-3'. PKC{zeta}-ODNs were designed as previously described to specifically hybridize with human PKC{zeta} mRNA at position 587–601 of the PKC{zeta} mRNA (45, 69). Procedures for PKC{zeta}-ODN treatment have been described previously (64) with the following modifications. Briefly, THP-1 cells (1 x 106 cells per treatment) were washed once with prewarmed PBS, resuspended with growth media (RPMI 1640 containing 2 mM L-glutamine + 10% FBS; 200 µl per treatment) without antibiotics in a 24-well plate. Lipofectamine (diluted to 6% in OPTI-MEM; 25 µl per treatment) was mixed with dilutions of ODNs or water (Mock) prepared in OPTI-MEM (25 µl per treatment) and incubated for 15 min at room temperature. Each mixture (50 µl) was then added to cells (1 x 106) in 200 µl of growth media for 3 h at 37 C with 5% CO2. Cells were then transferred to 4 ml of complete growth media (RMPI 1640 containing 2 mM L-glutamine + 10% FBS + 2 mM L-glutamine + 50 U/ml penicillin + 50 µg/ml streptomycin) and incubated for an additional 45 h. After 48 h, cells were used for total mRNA isolation or assayed for cAMP accumulation as described above. A portion of cells were washed once in ice-cold PBS and resuspended in cell lysis buffer (300 µl per well) for Western blot analysis.


   ACKNOWLEDGMENTS
 
We thank Dr. Barbara Sanborn for providing PHM1–31 cells, relaxin, and continued moral support. We also thank Drs. Jeff Frost, Andy Morris, and Mike Blackburn for advice and the use of microscopy equipment and Dr. Eric Neilson for providing MMC cells.


   FOOTNOTES
 
This work was supported by the Texas Advanced Research Program No. 011618-0059-1999 and the National Institutes of Health GM60419.

First Published Online December 16, 2004

Abbreviations: AC, Adenylyl cyclase; AMV, avian myeloblastosis virus; DAPI, 4',6-diamidino-2-phenylindole; DNase, deoxyribonuclease; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; LGR, leucine-rich repeat containing G protein-coupled receptor; LPS, lipopolysaccharide; MMC, mouse mesangial; ODN, oligodeoxynucleotide; PDE, phosphodiesterase; PDK, phosphoinositide-dependent kinase; PHM, pregnant human myometrial; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; PKC, protein kinase C; VEGF, vascular endothelial growth factor.

Received for publication July 8, 2004. Accepted for publication December 8, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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C. S. Samuel
Relaxin: Antifibrotic Properties and Effects in Models of Disease
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