S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.     

Transgenic overexpression of the Ca2+-binding protein S100A1 in the heart leads to increased in vivo myocardial contractile performance

S100A1, a Ca2+-sensing protein of the EF-hand family, is most highly expressed in myocardial tissue, and cardiac S100A1 overexpression in vitro has been shown to enhance myocyte contractile properties. To study the physiological consequences of S100A1 in vivo, transgenic mice were developed with cardiac-restricted overexpression of S100A1. Characterization of two independent transgenic mouse lines with approximately 4-fold overexpression of S100A1 in the myocardium revealed a marked augmentation of in vivo basal cardiac function that remained elevated after beta-adrenergic receptor stimulation. Contractile function and Ca2+ handling properties were increased in ventricular cardiomyocytes isolated from S100A1 transgenic mice. Enhanced cellular Ca2+ cycling by S100A1 was associated both with increased sarcoplasmic reticulum Ca2+ content and enhanced sarcoplasmic reticulum Ca2+-induced Ca2+ release, and S100A1 was shown to associate with the cardiac ryanodine receptor. No alterations in beta-adrenergic signal transduction or major cardiac Ca2+-cycling proteins occurred, and there were no signs of hypertrophy with chronic cardiac S100A1 overexpression. Our findings suggest that S100A1 plays an important in vivo role in the regulation of cardiac function perhaps through interacting with the ryanodine receptor. Because S100A1 protein expression is down-regulated in heart failure, increasing S100A1 expression in the heart may represent a novel means to augment contractility.     

Translocation of S100A1(1) calcium binding protein during heart surgery

Myocardial ischemia during cardiopulmonary bypass terminated by reperfusion generally leads to different degrees of damage of the cardiomyocytes induced by transient cytosolic Ca(2+) overload. Recently, much attention has been paid to the role of heart-specific Ca(2+)-binding proteins in the pathogenesis of myocardial ischemia-reperfusion injury. S100A1 is a heart-specific EF-hand Ca(2+)-binding protein that is directly involved in a variety of Ca(2+)-mediated functions in myocytes. The aim of our study was to investigate the localization and translocation of S100A1 in the human heart under normal (baseline) conditions and after prolonged ischemia and reperfusion of the myocardium. Our data suggest that S100A1 is directly involved in the transient perioperative myocardial damage caused by ischemia during open heart surgery in humans. Given its role in the contractile function of muscle cells, this S100 protein could be an important "intracellular link" in ischemia-reperfusion injury of the heart.     

The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway

The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dt_max), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I/R group. SAA exerts an anti-apoptotic role against myocardial IRI by inhibiting DUSP2-mediated JNK dephosphorylation and activating DUSP4/16-mediated ERK1/2 phosphorylation.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.     

Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis

The calcium-sensing receptor (CaSR) exists in many tissues, and its expression has been identified in rat cardiac tissue. However, the physiological importance and pathophysiological involvement of CaSR in homeostatic regulation of cardiac function are unclear. To investigate the relation of CaSR and apoptosis in cardiomyocytes, we examined the role of the CaSR activator gadolinium chloride (GdCl(3)) in rat neonatal ventricular cardiomyocytes. Expression of the CaSR protein was observed by Western blot. The apoptotic ratio of rat neonatal ventricular cardiomyocytes was measured with flow cytometry and immunofluorescence techniques. A laser scan confocal microscope was used to detect the intracellular concentration of calcium ([Ca(2+)](i)) in rat neonatal ventricular cardiomyocytes using the acetoxymethyl ester of fluo-3 (fluo-3/(AM)) as a fluorescent dye. The results showed that GdCl(3) increased the phosphorylation of extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal protein kinases (JNK), and p38. GdCl(3) also activated caspase 9 and increased apoptosis in myocyte by increasing [Ca(2+)](i). In conclusion, these results suggest that CaSR promotes cardiomyocyte apoptosis in rat neonatal ventricular cardiomyocytes through activation of mitogen-activated protein kinases and caspase 9 signaling pathways.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

ERK/PP1a/PLB/SERCA2a and JNK Pathways Are Involved in Luteolin-Mediated Protection of Rat Hearts and Cardiomyocytes following Ischemia/Reperfusion

Luteolin has long been used in traditional Chinese medicine for treatment of various diseases. Recent studies have suggested that administration of luteolin yields cardioprotective effects during ischemia/reperfusion (I/R) in rats. However, the precise mechanisms of this action remain unclear. The aim of this study is to confirm that luteolin-mediated extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways are responsible for their cardioprotective effects during I/R. Wistar rats were divided into the following groups: (i) DMSO group (DMSO); (ii) I/R group (I/R); (iii) luteolin+I/R group (Lut+I/R); (iv) ERK1/2 inhibitor PD98059+I/R group (PD+I/R); (v) PD98059+luteolin+I/R group (PD+Lut+I/R); and (vi) JNK inhibitor SP600125+I/R group (SP+I/R). The following properties were measured: contractile function of isolated heart and cardiomyocytes; infarct size; the release of lactate dehydrogenase (LDH); the percentage of apoptotic cells; the expression levels of Bcl-2 and Bax; and phosphorylation status of ERK1/2, JNK, type 1 protein phosphatase (PP1a), phospholamban (PLB) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Our data showed that pretreatment with luteolin or SP600125 significantly improved the contraction of the isolated heart and cardiomyocytes, reduced infarct size and LDH activity, decreased the rate of apoptosis and increased the Bcl-2/Bax ratio. However, pretreatment with PD98059 alone before I/R had no effect on the above indexes. Further, these consequences of luteolin pretreatment were abrogated by co-administration of PD98059. We also found that pretreatment with PD98059 caused a significant increase in JNK expression, and SP600125 could cause ERK1/2 activation during I/R. In addition, we are the first to demonstrate that luteolin affects PP1a expression, which results in the up-regulation of the PLB, thereby relieving its inhibition of SERCA2a. These results showed that luteolin improves cardiomyocyte contractile function after I/R injury by an ERK1/2-PP1a-PLB-SERCA2a-mediated mechanism independent of JNK signaling pathway.     

Epidermal growth factor potentiates cholecystokinin/gastrin receptor-mediated Ca2+ release by activation of mitogen-activated protein kinases

Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+](i)) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+](i) that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1-17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK(2)R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK(2)R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK(2)R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+](i) in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK(2)R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK(2)R-regulated signaling pathways.     

The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.     

RGD-containing peptides activate S6K1 through beta3 integrin in adult cardiac muscle cells

The enzyme p70S6 kinase (S6K1) is critical for cell growth, and we have reported its activation during cardiac hypertrophy. Because cardiac hypertrophy also involves integrin activation, we analyzed whether integrins could contribute to S6K1 activation. Using adult feline cardiomyocytes, here we report that integrin-interacting Arg-Gly-Asp (RGD) peptides activate S6K1 as observed by band shifting, kinase activity and phosphorylation at Thr-389 and Thr-421/Ser-424 of S6K1, and S6 protein phosphorylation. Perturbation of specific integrin function with blocking antibodies and by overexpressing the beta1A cytoplasmic tail revealed that beta3 but not beta1 integrin mediates the RGD-induced S6K1 activation. This activation is focal adhesion complex-independent and is accompanied by the activation of extracellular signal-regulated kinases 1/2 (ERK) and mammalian target of rapamycin (mTOR). Studies using specific inhibitors and dominant negative c-Raf expression in cardiomyocytes indicate that the S6K1 activation involves mTOR, MEK/ERK, and phosphatidylinositol 3-kinase pathways and is independent of protein kinase C and c-Raf. Finally, addition of fluorescent-labeled RGD peptide to cardiomyocytes exhibits its internalization and localization to the endocytic vesicles, and pretreatment of cardiomyocytes with endocytic inhibitors reduced the S6K1 activation. These data suggest that RGD interaction with beta3 integrin and its subsequent endocytosis trigger specific signaling pathway(s) for S6K1 activation in cardiomyocytes and that this process may contribute to hypertrophic growth and remodeling of myocardium.     

S100A1 decreases calcium spark frequency and alters their spatial characteristics in permeabilized adult ventricular cardiomyocytes

S100A1, a Ca2+-sensor protein of the EF-hand type, exerts positive inotropic effects in the heart via enhanced cardiac ryanodine receptor (RyR2) activity. Here we report that S100A1 protein (0.1microM) interacts with the RyR2 in resting permeabilized cardiomyocytes at free Ca2+-levels comparable to diastolic Ca2+-concentrations ( approximately 150nM). Alterations of RyR2 function due to S100A1 binding was assessed via analysis of Ca2+-spark characteristics. Ca2+-spark frequency, amplitude and duration were all reduced upon perfusion with 0.1microM S100A1 protein by 38%, 14% and 18%, respectively. Most likely, these effects were conveyed through the S100A1 C-terminus (S100A1-ct; amino acids 75-94) as the corresponding S100A1-ct peptide (0.1microM) inhibited S100A1 protein binding to the RyR2 and similarly attenuated frequency, amplitude and duration of Ca2+-sparks by 52%, 8% and 26%, respectively. Accordingly, the sarcoplasmic reticulum (SR) Ca2+-content was slightly increased but the stoichiometry of other accessory RyR2 modulators (sorcin/FKBP12.6) remained unaltered by S100A1. Hence, we propose S100A1 as a novel inhibitory modulator of RyR2 function at diastolic Ca2+-concentrations in rabbit ventricular cardiomyocytes.     

H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.