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Muscarinic acetylcholine receptors induce the expression of the immediate early growth regulatory gene CYR61.

In brain, muscarinic acetylcholine receptors (mAChRs) modulate neuronal functions including long term potentiation and synaptic plasticity in neuronal circuits that are involved in learning and memory formation. To identify mAChR-inducible genes, we used a differential display approach and found that mAChRs rapidly induced transcription of the immediate early gene CYR61 in HEK 293 cells with a maximum expression after 1 h of receptor stimulation. CYR61 is a member of the emerging CCN gene family that includes CYR61/CEF10, CTGF/FISP-12, and NOV; these encode secretory growth regulatory proteins with distinct functions in cell proliferation, migration, adhesion, and survival. We found that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Stimulation of mAChRs induced CYR61 expression in primary neurons and rat brain where CYR61 mRNA was detected in cortical layers V and VI and in thalamic nuclei. In contrast, CTGF and NOV expression was not altered by mAChRs neither in neuronal tissue culture nor rat brain. Receptor subtype analyses demonstrated that m1 and m3 mAChR subtypes strongly induced CYR61 expression, whereas m2 and m4 mAChRs had only subtle effects. Increased CYR61 expression was coupled to mAChRs by both protein kinase C and elevations of intracellular Ca(2+). Our results establish that CYR61 expression in mammalian brain is under the control of cholinergic neurotransmission; it may thus be involved in cholinergic regulation of synaptic plasticity.

In brain, muscarinic acetylcholine receptors (mAChRs) modulate neuronal functions including long term potentiation and synaptic plasticity in neuronal circuits that are involved in learning and memory formation. To identify mAChR-inducible genes, we used a differential display approach and found that mAChRs rapidly induced transcription of the immediate early gene CYR61 in HEK 293 cells with a maximum expression after 1 h of receptor stimulation. CYR61 is a member of the emerging CCN gene family that includes CYR61/CEF10, CTGF/FISP-12, and NOV; these encode secretory growth regulatory proteins with distinct functions in cell proliferation, migration, adhesion, and survival. We found that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Stimulation of mAChRs induced CYR61 expression in primary neurons and rat brain where CYR61 mRNA was detected in cortical layers V and VI and in thalamic nuclei. In contrast, CTGF and NOV expression was not altered by mAChRs neither in neuronal tissue culture nor rat brain. Receptor subtype analyses demonstrated that m1 and m3 mAChR subtypes strongly induced CYR61 expression, whereas m2 and m4 mAChRs had only subtle effects. Increased CYR61 expression was coupled to mAChRs by both protein kinase C and elevations of intracellular Ca 2؉ . Our results establish that CYR61 expression in mammalian brain is under the control of cholinergic neurotransmission; it may thus be involved in cholinergic regulation of synaptic plasticity.
The cholinergic system in mammalian brain is involved in higher cognitive functions including attention, learning, and memory (1). A major source of the neurotransmitter acetylcholine are cholinergic neurons of the basal forebrain that innervate neurons throughout the cerebral cortex, the hippocampus, the amygdala, and some thalamic nuclei (2,3). The degenera-tion of cholinergic neurons in the basal forebrain is associated with Alzheimer's disease; lesions within this brain region, as well as mAChR 1 antagonists, impair cognitive functions in mammals (4 -7). Muscarinic AChRs are G protein-coupled cell surface receptors with seven transmembrane topologies (8). Five different subtypes are expressed in different brain regions on pre-and postsynaptic neuronal compartments (9). They have excitatory and inhibitory effects on cholinergic synapses by modulating the conductance of K ϩ and Ca 2ϩ ion channels (10 -12) and by coupling to several intracellular second messengers as follows: mAChR subtypes m1, m3, and m5 activate protein kinase C (PKC) (13,14), increase intracellular Ca 2ϩ and cAMP (13)(14)(15), stimulate the MAP kinase pathway (16,17), and activate the phospholipases A 2 (18) and D (19). In contrast, m2 and m4 mAChRs inhibit adenylyl cyclase (13,14,16,17,20,21).
The storage of long term memory is associated with the generation of long term potentiation and with changes in synaptic plasticity that are mediated by alterations in the expression of activity-dependent genes (22). These include immediate early transcription factors and activity-dependent genes with potential functions in modulating long term memory by structural modifications of preexisting synapses, in the generation of new neuronal connections, as well as in neurite extension and dendritic ramification (23)(24)(25)(26)(27). Cholinergic mechanisms are involved in learning and memory processes in that mAChRs promote the generation of long term potentiation (28) and activate the expression of the transcription factors c-fos, jun-B, Egr-1, Egr-2, Egr-3, and Egr-4 (29,30).
To identify mAChR-inducible genes, we established a differential display screen of genes activated in response to mAChR stimulation with the acetylcholine analog carbachol. We identified the immediate early gene CYR61 that encodes an extracellular signaling molecule to be differentially regulated by mAChRs. CYR61 belongs to the emerging CCN gene family (CYR61/CEF10, CTGF/FISP-12, NOV, ELM-1, COP-1, and WISP-3) of secreted growth regulatory proteins that bind to components of the extracellular matrix and the cell surface (31,32). CYR61 and CTGF (connective tissue growth factor) are both growth factor-inducible immediate early genes in fibroblasts (33,34), whereas the protooncogene NOV (nephroblastoma overexpressed), involved in Wilms tumor, is expressed in quiescent cells and down-regulated after serum stimulation (35,36). Consistent with their expression kinetics, secreted CYR61 and CTGF proteins exhibit growth-promoting cellular functions (37,38), whereas the overexpression of NOV protein has growth-inhibitory effects and is associated with normal differentiation of the central nervous system and kidney in general (35,39,40). CTGF gene is a downstream target of TGF-␤, mediating TGF-␤-related actions in connective tissue cells during wound healing and skin disorders (41,42), and both proteins, CYR61 and CTGF, are regulators of angiogenesis, mediating cellular adhesion and migration of endothelial cells in an integrin-␣ v ␤ 3 -dependent manner (43,44). Even though CYR61, CTGF, and NOV are expressed in the developing nervous system (40,45), less is known about their function in adult brain.
We provide evidence that CYR61 and its family members CTGF and NOV are expressed throughout the mammalian brain and that transcription of CYR61, but not that of CTGF and NOV, is under the control of mAChR signaling in primary cortical neurons and in mammalian brain.
Cortical Neurons-Primary cell cultures from embryonic day 18 rat brains were prepared according to the protocol from Segal and Manor (47). In brief, cerebral cortices were dissected in chilled Hanks' balanced salt solution (Life Technologies, Inc.) and mechanically dissociated with a fire-polished Pasteur pipette in small volumes of Hanks' balanced salt solution. Dissociated cells were suspended in 10% horse serum and minimal essential medium enriched with 0.6% glucose and were plated onto poly-L-lysine-coated glass coverslips in 6-well plates, at a density of about 10 6 cells per well. After 3 h, plating media were replaced by minimal essential medium enriched with Earle's salts, 0.6% glucose, and Bottenstein's N 2 supplements. In order to block the proliferation of glial cells, 3-4 days after plating media were exchanged for growth media supplemented with 0.5 M cytosin-␤-arabinofuranoside HCl (AracC). Thereafter, growth media were exchanged once a week. After 9 days in culture, subconfluent layers of primary cells were subjected to drug treatments, followed by RNA preparation or measurements of phosphoinositide turnover (48). The content of glial cells was estimated by immunohistochemistry with antibodies against glial fibrillary acidic protein and compared with neuronal staining with an antibody against tau, and it revealed that consistently more than 70% of the cultured cells had a neuronal phenotype.
RNA Preparation and Reverse Transcription-Total RNA was prepared by using either the RNeasy Kit (Qiagen) or Trizol reagent (Life Technologies, Inc.) following the manufacturer's instructions. RNA was stored at Ϫ80°C.
The RNA preparations from fetal rat lung (P1), adult rat thyroid, spleen, and brain were treated with DNase I (Roche Molecular Biochemicals) together with RNasin (Promega) for 30 min, followed by phenol protein extraction, and ethanol precipitation. 0.2 g of RNA preparation was transcribed to cDNA by using the SuperScript TM II ReverseTranscriptase (Life Technologies, Inc.) with equal amounts of one base anchor primers HT 11 A (5ЈTGCCGAAGCTTTTTTTTTTTA3Ј), HT 11 C (5ЈTGCCGAAGCTTTTTTTTTTTC3Ј), and HT 11 G (5ЈTGC-CGAAGCTTTTTTTTTTTG3Ј).
Differential Display-The differential display approaches were performed as described in detail by von der Kammer et al. (49). h-CYR61 was identified as a differentially expressed gene by using the random primer DD20 (5ЈTGCCGAAGCTTTGGTCAT3Ј) or DD32 (5ЈTGCCGA-AGCTTGGAGCTT3Ј) together with the anchor primer HT 11 A (5ЈTGC-CGAAGCTTTTTTTTTTTA3Ј).
Northern Blotting-5-10 g of total RNA were separated in 1% formaldehyde-containing agarose gels, and the RNA was blotted onto nylon membranes (Hybond-N ϩ , Amersham Pharmacia Biotech). Membranes were hybridized with [␣-32 P]dCTP (NEN Life Science Products)labeled cDNA probes that were generated by using the Megaprime DNA labeling kit (Amersham Pharmacia Biotech). Membranes were washed under high stringency conditions, and x-ray films were exposed for 1-72 h. To control for equal loading of RNA, the identical membranes were probed with a 700-bp cDNA fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or with a ␤-actin cDNA fragment (CLONTECH).
Construction of a m1 mAChR-stimulated ZAP Express cDNA Library-HEK 293 m1 mAChR cells were stimulated with carbachol for 1 h, and poly(A ϩ ) mRNA was prepared from total RNA by using the Poly(A)TTract IV kit (Promega). cDNA synthesis from poly(A ϩ ) mRNA was performed following a modified ZAP-cDNA synthesis protocol (Stratagene), and cDNA cloning into ZAP Express vector arms as well as preparation of the cDNA library were done according to the manufacturer's instructions (Stratagene). A library screen of 4 ϫ 10 4 clones was performed with a radiolabeled human CYR61 cDNA probe following the ZAP Express protocol (Stratagene).
Human CYR61, CTGF, and NOV cDNA Fragments for Northern Blots and Library Screening-We amplified a 1403-bp h-CYR61 cDNA fragment by using cDNA from carbachol-stimulated (1 h) HEK 293 m1 mAChRs cells. The forward primer EMHF (5ЈCCAAAACGGGGAAAG-TTTCCAGCC3Ј) derived from the partial human sequence A33210 (EMBL accession number Z50168) that showed homology to the 5Ј end of murine cyr61. The reverse primer EMHR (5ЈTTCCAGTATTACATT-TCCCCTCCC3Ј) was homolog to the 3Ј end of h-CYR61 identified by our differential display experiment. The PCR products were separated by agarose gel electrophoresis, purified, and cloned into the SmaI restriction site of pBluescript KS (Stratagene). The cloned PCR product was sequenced, and a PstI/BamHI restriction fragment that contained 1403 bp was used as a probe both for screening a ZAP Express cDNA library and for probing Northern blots. Human cDNA clones of CTGF (h-CTGF) and NOV (NOVH) were obtained as expressed sequence tag from the Research Center of the German Genome Project at the Max Planck Institute for Molecular Genetics in Berlin, Germany (RZPD) (50). A 1678-bp EcoRI/XhoI fragment from human CTGF clone (IMAGp998P101426), and a 1735-bp HindIIIINotI fragment from human NOV clone (IMAGp998J07280) were used to generate radioactive probes for hybridization.
Rat CYR61, CTGF, and NOV cDNA Fragments for Northern Blots and Library Screening-A 1266-bp r-CYR61 cDNA fragment and a 881-bp r-CTGF cDNA fragment were amplified by PCR using fetal rat lung cDNA as template together with the primer combinations rCYR61Ef (5ЈTCTGAAAGGGATCTGCAGAGCTCA3Ј) and rCYR61Er (5ЈGGCTTTCACTTGACCCAA CTAATC3Ј) or rCTGF1f (5ЈTGACTGC-CCCTTCCCGAGAAGGGT3Ј) and rCTGF4r (5ЈCCTTAGTGTGCGT-TCTGTCACTGT3Ј). A 947-bp NOVR cDNA fragment was amplified by using adult rat brain cDNA as template together with the forward primer NOVM1f (5ЈTCGATGGGGTCATTTACCGCAACG3Ј) and reverse primer NOVM4r (5ЈCATATCCTGAGCTTACCTTGATTC3Ј). All PCR products were separated by agarose gel electrophoresis, purified, cloned into the pCR-BluntII-TOPO vector (Invitrogen), and sequenced.
Rat CYR61 Sequence-A complete r-CYR61 cDNA sequence was identified by sequence analysis of overlapping cDNAs from different tissues. Rat lung and placenta cDNA libraries (numbers 614 and 618) obtained from the Research Center of the German Genome Project at the Max Planck Institute for Molecular Genetics in Berlin (RZPD) (50) were screened with a 1266-bp r-CYR61 cDNA fragment following RZPD instructions. Two lung cDNA clones (p614K0171Q2 and p614O2448Q2) were identified that displayed r-CYR61 5Ј end (nt 1-534) as well as one placenta cDNA clone p618K0827Q2 corresponding to 3Ј end (nt 1016 -1987). PCR products that included the missing region from nt 443-1088 were generated from rat lung, thyroid, and spleen cDNAs of two different animals by using the primers rCYR61Ef (5ЈTCTGAAAGGGATCT-GCAGAGCTCA3Ј) and rCYR61R1 (5ЈGCATCCTGCATAAGTAAATC-GGAC3Ј).
In Vivo Experiments-Adult male Harlan Sprague-Dawley rats were injected intraperitoneally with the mAChR agonist pilocarpine (25 mg/ kg; Sigma) or with the vehicle PBS as an injection control. Animal behavior was monitored until the animals were decaptured 75 min after drug application. Brains were rapidly dissected and frozen on dry ice. In Situ Hybridization-Rat CYR61 (nt 928 -1523), r-CTGF (nt 644 -1524), or NOVR (nt 224 -1280) cDNA fragments were subcloned into pCR-BluntII-TOPO vector (Invitrogen). Plasmid DNA was digested by restriction enzymes BamHI or XhoI, separated by agarose gel electrophoresis, purified, and subjected to phenol protein extraction and ethanol precipitation. Linearized plasmid DNA was used as template to generate radiolabeled sense and antisense cRNA probes by using either T7 or SP6 RNA polymerase (Roche Molecular Biochemicals) together with [␣-35 S]UTP (NEN Life Science Products), GTP, ATP, and CTP, dithiothreitol, and RNase Out (Roche Molecular Biochemicals) for 2 h at 37°C. Unincorporated nucleotides were removed by ProbeQuant G50 gel filtration (Amersham Pharmacia Biotech). Rat brains were cut on a Leica cryostat microtome in 16-m slices, thawed on glass slides, and stored at Ϫ80°C before use. The slices were fixed in 4% paraformaldehyde, acetylated, and prehybridized for 3 h at 55°C with prehybridization buffer (50% formamide, 5ϫ hybridization salts, 5ϫ Denhardt's, 0.2% SDS, 10 mM dithiothreitol, 250 g/ml salmon sperm DNA (Stratagene), 250 g/ml yeast tRNA (Roche Molecular Biochemicals)) in a humidified chamber. Hybridization was performed for 18 h at 55°C with radiolabeled cRNA probes (5000 cpm/l) in prehybridization buffer containing 10% dextran sulfate. The slices were dehydrated, washed under non-stringent conditions in 4ϫ SSC at room temperature, followed by RNase A treatment. Two stringent wash steps were performed in 2ϫ SSC for 10 min at 55°C. The slices were dehydrated, and a x-ray film (Kodak Biomax MR) was exposed for 3-14 days. Emulsion dips were prepared with NTB-2 photoemulsion (Kodak), exposed for 1-4 weeks, developed, and fixed. Sections were counterstained with Mayers Hä malaun (Merck) (51).

Identification of Human CYR61 by Differential Display-By
using a differential display screen of mRNAs expressed in response to stimulation of m1 mAChR expressing HEK 293 cells, we identified two redundant bands of 450 and 540 bp in length, which were absent in parallel fractions of unstimulated cells (data not shown). Either band contained cDNA corresponding to human CYR61 (h-CYR61), a homolog of the murine growth factor-inducible immediate early gene cyr61 (33). To verify that mAChRs are coupled to the transcriptional activation of the h-CYR61 gene, we compared RNA from m1 mAChRexpressing cells that were stimulated with carbachol for different incubation times with unstimulated m1 mAChRexpressing cells. Northern blot analysis showed no detectable levels of h-CYR61 mRNA in unstimulated cells; h-CYR61 signal increased 15 min after stimulation, attained a maximum within 50 -60 min, and decreased to lower but higher than control levels until 240 min after stimulation (Fig. 1A). An increase of h-CYR61 expression was also detectable in HEK 293 m1 cells that were treated with carbachol in the presence of the protein synthesis inhibitor cylcoheximide (Fig. 1B).
Human and Rat CYR61 Sequences-We identified a human CYR61 full-length cDNA clone by screening a ZAP Express cDNA library prepared from carbachol-stimulated m1 mAChRexpressing HEK 293 cells. By using a 1403-bp h-CYR61 cDNA probe that was obtained by PCR with cDNA from carbacholstimulated HEK 293 m1 mAChR cells, we found that 7 out of 40,000 clones were human homologues of the murine cyr61 gene. The sequence of a 2021-bp human CYR61 clone encoding a complete open reading frame predicted a 381-amino acid protein. The sequence data of our h-CYR61 full-length clone were submitted to GenBank TM /EBI Data Bank (accession number Y12084), and they confirm the h-CYR61 sequence that was recently published by Jay et al. (52).
Three different types of cDNA sources were used to identify the complete rat CYR61 (r-CYR61) cDNA sequence (Gen-Bank TM accession number AF218568). By screening a rat lung cDNA library offered by the Research Center of the German Genome Project (50) with a 1266-bp r-CYR61 cDNA probe generated by PCR from fetal rat lung, two lung cDNA clones (p614K0171Q2 and p614O2448Q2) were obtained that contained the 5Ј end of r-CYR61 (nt 1-534). PCR products that were generated from rat lung, thyroid, and spleen cDNA corresponded to nt 443-1088. Finally, a cDNA clone (p618K0827Q2) corresponding to the 3Ј end of r-CYR61 (nt 1016 -1987) was identified by screening a rat placenta cDNA library from RZPD. The consensus sequence of 1987-bp includes one open reading frame encoding a predicted protein of 379 amino acids. The human and rat CYR61 protein sequences were 93% identical. Characteristic features of the predicted human and rat CYR61 proteins included N-terminal signal peptides, high content of conserved cysteines, and several domains that are typical for members of the CCN (CYR61/ CEF10, CTGF/FISP-12/, NOV, ELM-1, WISP-3 and COP-1) family (31,32) as follows: an insulin-like growth factor-binding protein domain, a von Willebrand factor type C motif for possible oligomerization, a thrombospondin-1 domain that can bind both soluble and extracellular matrix molecules, and a C-terminal domain for putative dimerization.
Expression of CYR61, CTGF, and NOV in Human Brain-CYR61 and its relatives CTGF and NOV were the first genes that were identified to be members of the CCN gene family (31). Even though these genes are characterized in peripheral tissues, less is known about their function in brain. In order to demonstrate the expression of CYR61, CTGF, and NOV in human brain, we probed Northern blots of poly(A ϩ ) RNA from several regions of adult human brains with cDNA probes of these three family members and found all of them to be expressed throughout human brain (Fig. 2). The strongest expression of h-CYR61 occurred in spinal cord, frontal, temporal, and occipital cortices, as well as in the hippocampus and caudate nucleus. In addition to the expected message of 2.4 kilobase pairs we detected a 3.8-kilobase pair message that may represent a splice variant of h-CYR61 mRNA. The expression from HEK 293 m1 mAChR cells that were treated for 1 h with carbachol alone or together with the protein synthesis inhibitor cycloheximide was probed with a radiolabeled 1403-bp h-CYR61 cDNA fragment. Cycloheximide was applied to the cells 15 min before carbachol treatment. To control for equal RNA loading, the blots were probed with a specific GAPDH cDNA. ctr, untreated HEK 293 m1 mAChR cells; CCh, carbachol; Chx, cycloheximide. X-ray film exposed to Northern blot: h-CYR61, 48 h; GAPDH, 1 h; kb, kilobase pairs. pattern of h-CTGF was similar to that of h-CYR61 except that tissue levels of h-CTGF transcripts were lower in hippocampus, caudate nucleus, and corpus callosum (Fig. 2). NOVH was expressed at high levels in amygdala and spinal cord, as well as in brain cortex and hippocampus (Fig. 2).
Muscarinic AChRs Induce the Expression of r-CYR61 in Primary Neurons-To investigate r-CYR61, r-CTGF, and NOVR expression in neuronal cells that endogenously express mAChRs, we stimulated a mixed culture of primary cortical and hippocampal neurons from embryonic (E18) rat brains with carbachol for 1 or 3 h (Fig. 3A). We found all three genes to be constitutively expressed. The stimulation with carbachol for 1 h increased basal r-CYR61 mRNA levels 3.5-fold, and this increase was blocked by atropine (Fig. 3, A and B). After 3 h of carbachol treatment r-CYR61 expression decreased to basal levels. In contrast, r-CTGF and NOVR expression was not influenced by mAChR stimulation, nor after 1 or 3 h of carbachol treatment (Fig. 3, A and B). The inhibition of m2 mAChRs with the m2-antagonist gallamine together with carbachol failed to block the carbachol-induced r-CYR61 expression (data not shown).
mAChR-dependent Regulation of CYR61 in the Mammalian Brain-In the mammalian brain, neuronal cells in the cortex, the hippocampus, and some thalamic nuclei receive cholinergic input from the basal forebrain (2, 3). To investigate whether mAChR regulate CYR61 expression in vivo, the mAChR agonist pilocarpine was administered to adult rats and brains were prepared 75 min later. Brains of non-injected and PBS-injected animals were used as controls. In situ hybridization analyses of cryostat slices of forebrain tissue from control and pilocarpinetreated animals demonstrated that expression of r-CYR61, but not r-CTGF or NOVR readily increased in response to mAChR stimulation (Fig. 4A). In both non-injected and PBS-injected control animals, we detected a very low basal expression of r-CYR61 that increased after pilocarpine treatment in cortical layer VI as well as in some cells of cortical layer V and in thalamic nuclei (Fig. 4, A and B). Rat CTGF mRNA was predominantly expressed in cortical layer VI in both control and pilocarpine-treated rats (Fig. 4A). Comparable to r-CTGF, NOVR expression was not altered by mAChR stimulation. NOVR mRNA was detected in cortical layers II/III, V, and VI, the pyramidal cell CA1-CA3 hippocampal regions, and the amygdala (Fig. 4A).
Muscarinic AChR Subtypes m1-m4 Induced Transcription of Human CYR61-To determine which mAChR subtype can couple to h-CYR61 expression, we analyzed Northern blots of RNA obtained from carbachol-treated HEK 293 cells stably expressing m1, m2, m3, or m4 mAChRs as well as untransfected wild type cells. h-CYR61 expression was differently affected by these receptor subtypes (Fig. 5A). m1 and m3 mAChRs increased cellular levels of h-CYR61 mRNA 5-10 times as compared with unstimulated control cells (Fig. 5B). In contrast, m2 and m4 mAChRs-expressing cells stimulated h-CYR61 expression only weakly. In carbachol-stimulated as well as in unstimulated wild type HEK 293 cells minimal h-CYR61 signals were detected on Northern blots.
Multiple Internal and External Signals Coupled CYR61 Expression to mAChRs in HEK 293 Cells-To determine the signal transduction pathways that couple CYR61 induction to mAChRs, we treated HEK 293 m1 mAChRs cells with activa-FIG. 2. Expression of CCN genes in human brain. The CCN gene family members CYR61, CTGF, and NOV were expressed in adult human brain regions. Human brain Northern blots loaded with poly(A ϩ ) RNA (CLONTECH) were hybridized with a radiolabeled 1403-bp h-CYR61 cDNA probe, a 1678-bp h-CTGF cDNA probe, and a 1735-bp NOVH cDNA probe. The h-CTGF and NOVH probes were derived from human expressed sequence tags obtained from the Resource Center of the German Human Genome Project at the Max-Planck-Institute for Molecular Genetics, Berlin (50). As a loading control, both blots were probed with a human ␤-Actin cDNA fragment (CLONTECH). X-ray films exposed to Northern blots: h-CYR61, 48 h; NOVH, 24 h; h-CTGF, 72 h; ␤-Actin, 1 h. kb, kilobase pairs.

FIG. 3. Muscarinic mAChRs induce CYR61 expression in rat primary neurons.
Rat CYR61, r-CTGF, and NOVR expression in a mixed culture of primary cortical and hippocampal neurons after carbachol treatment are shown. A, Northern blots with 10 g of total RNA from rat primary neurons treated with mAChR agonist carbachol (CCh) for 1 or 3 h alone or together with mAChR antagonist atropine (Atr) were hybridized with a r-CYR61, r-CTGF, and NOVR probe. Atropine was applied to the cells 10 min before carbachol treatment. GAPDH was used as a loading control. X-ray film exposed to Northern blots: r-CYR61, 48 h; r-CTGF, 24 h; NOVR, 12 h; GAPDH, 1 h. B, densitometric analyses and quantification of r-CYR61, r-CTGF, and NOVR mRNA expression in primary neurons normalized to cellular levels of GAPDH mRNA. kb, kilobase pairs. tors or inhibitors of known signaling cascades. Alternatively, cells were treated with the mAChR agonist carbachol together with pharmacological inhibitors. Northern blots of RNA derived from these cells were hybridized with a h-CYR61 cDNA probe (Fig. 6A). The muscarinic antagonist atropine blocked the carbachol-induced increase in cellular levels of h-CYR61 mRNA in HEK 293 m1 cells. Stimulation of PKC with the phorbol ester phorbol myristate acetate increased h-CYR61 expression 3.0-fold (Fig. 6B), and this increase was blocked by the PKC inhibitor GF109203X. Inhibition of PKC with GF109203X along with carbachol stimulation, however, still led to transcriptional activation of h-CYR61 but to lower magnitudes as compared with carbachol alone. These results demonstrate that stimulation of PKC is sufficient but not necessary to induce h-CYR61 expression. In contrast, the activation of protein kinase A (PKA) by 8-bromo-cAMP failed to induce h-CYR61 transcription, and the PKA inhibitor H7 failed to block the carbachol-induced increase in CYR61 message. More-over, activation of phospholipase A 2 by mellitin increased h-CYR61 expression, and this effect was slightly decreased by the phospholipase A 2 inhibitor DEDA, but DEDA failed to block the carbachol-induced increase. Both the increase of intracellular Ca 2ϩ levels by ionomycin as well as serum induced h-CYR61 expression. DISCUSSION In brain, mAChR are associated with long term memory formation that depends on changes in synaptic plasticity, including modifications of preexisting synapses and the generation of new neuronal connections. These synaptic modifications are mediated by alterations in the expression of activity-dependent genes. Muscarinic AChR induce the expression of immediate early gene transcription factors c-fos, jun-B, Egr-1/ Krox20, and Egr-2/Krox24 (53), and a variety of activitydependent genes are known that proteins directly influence synaptic plasticity, like growth factors (␤-activin, Ref.  (32), but their function in brain is still unknown. The results of this study show that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Among these three family members, the immediate early gene CYR61 was unique in that its expression was regulated by mAChR activity, both in primary cortical neurons and in adult rat brain.

FIG. 4. Muscarinic mAChRs induce the expression of r-CYR61
in rat brain. In situ hybridization experiments with rat brains were performed with rats that were injected intraperitoneally with the mAChR agonist pilocarpine (25 mg/kg). A, frontal sections of control and rat brains stimulated for 75 min were hybridized with r-CYR61, r-CTGF, or NOVR antisense riboprobes. Rat CYR61 revealed low basal expression in control animals but was strongly induced in cortical layers V, VI, and thalamic nuclei (thal) in pilocarpine-treated rats. Rat CTGF and NOVR expression was not altered by mAChR stimulation as their mRNA distribution did not differ in control and pilocarpinetreated rat brain. R-CTGF was predominantly expressed in cortical layer VI. NOVR mRNA was detected in cortical layers II/III, V, VI, the hippocampal CA1-3 region, and the amygdala (ad). X-ray films exposed to brain slices: r-CYR61, 14 days; r-CTGF, 14 days; NOVR, 3 days. B, rat CYR61 in cortical layer VI of pilocarpine-treated animals compared with corresponding region of control rats. VI, cortical layer VI; CC, corpus callosum; arrows indicate r-CYR61 positive grains.

FIG. 5. Muscarinic receptor subtypes differently induce
h-CYR61 expression. HEK 293 wild type (wt) cells or cells either transfected with m1, m2, m3, or m4 mAChRs were treated with or without 1 mM carbachol (CCh) for 1 h. A, Northern blot containing total RNA (5 g per lane) was hybridized with a 1403-bp h-CYR61 cDNA probe. GAPDH probe was used as a loading control. X-ray film exposed to Northern blot: h-CYR61, 48 h; GAPDH, 1 h. B, quantification of h-CYR61 expression normalized to GAPDH control. Bars represent means derived from two representative independent stimulation and hybridization experiments. kb, kilobase pairs. In order to identify genes that expression is regulated in response to mAChR stimulation, we established a differential display screen by comparing gene expression of unstimulated and carbachol-stimulated HEK 293 cells that stably overexpress the m1 mAChR subtype (49). HEK 293 cells are human embryonic kidney cells that do not express mAChRs endogenously. It has been shown that recombinant expressed mAChR subtypes in HEK 293 cells are coupled to the same signal transduction pathways as in neuronal cells and mediate mAChR-related processing of the amyloid precursor protein in the same manner as in central nervous system (13,14,46,59). In addition to that the expression of the immediate early transcription factor genes Egr-1/Krox24 and Egr-2/Krox20 is induced by mAChR stimulation in HEK 293 m1 cells as well as in brains from rats that were treated with the mAChR agonist pilocarpine (29,30,53).
In unstimulated HEK 293 m1 mAChRs cells, the acetylcholine analog carbachol rapidly induced h-CYR61 expression within 15 min of stimulation. h-CYR61 expression reached a maximum 50 -60 min after stimulation and attained lower but clearly detectable levels up to 4 h after stimulation. This time course was consistent with the kinetics of serum induction of murine cyr61 expression in fibroblasts (33,60). In addition carbachol-mediated h-CYR61 expression increased in the presence of the protein synthesis inhibitor cycloheximide, proving CYR61 to be an immediate early gene that expression is independent of de novo protein synthesis in response to mAChRrelated signaling.
Consistent with the fact that CYR61, CTGF, and NOV are expressed during development of the central nervous system (40,45), we found basal levels of message for these three genes in primary cortical neurons prepared from E18 embryonic brains. Carbachol-induced stimulation of r-CYR61 of endogenously expressed mAChRs in primary cortical neurons was atropine-sensitive, indicating that muscarinic receptors mediated the activation of r-CYR61 expression. The failure of the m2-antagonist gallamine to block this response indicated that it was primarily mediated by m1, and possibly by m3 mAChRs. Receptor subtype analyses in HEK 293 cells underscored that finding in that m1 and m3 rather than m2, and m4 mAChRmediated signaling induced the expression of CYR61. The G q / G 11 -coupled m1 and m3 receptors strongly induced h-CYR61 expression. In contrast, the G i /G o -coupled m2 and m4 mAChRs only lead to a very subtle induction of h-CYR61 that was still higher, however, than the absent response in untransfected control cells. This subtle increase may be related to the weak stimulation of PKC coupled to m2 and m4 mAChRs (14,20). Consistent with our finding that mainly m1 and m3 mAChR subtypes induce CYR61 expression in receptor-transfected cells, we found two principal signaling mechanisms that may couple h-CYR61 expression to mAChR activation, PKC and Ca 2ϩ . These signal transduction mediators independently increased cellular levels of h-CYR61 mRNA, but our data do not exclude the possibility of cross-talk among them. Recent studies show that the expression of CYR61 is activated during the basic fibroblast growth factor-induced differentiation process in the embryonic hippocampal neuronal cell line H19 -7 in a MAP kinase-dependent and -independent manner (61). In addition to PKC and Ca 2ϩ , muscarinic m1 receptors are known to activate MAP kinase signaling (62,63) strongly suggesting that MAP kinase signaling may couple muscarinic receptor stimulation to CYR61 expression. To exclude that mAChR-dependent increases in cAMP or PKA regulate h-CYR61 expression, we treated cells with 8-bromo-cAMP, and we found that it failed to change basal levels of h-CYR61 mRNA. Likewise, inhibition of PKA with H7 failed to block the carbachol-induced stimulation of h-CYR61 expression. These data argue against a significant role of cAMP-PKA-CREB1 signaling in coupling h-CYR61 expression to mAChR in HEK 293 cells. Taken together our data are compatible with the concept that mAChRs induce h-CYR61 expression via PKC, MAP kinase, and Ca 2ϩ signaling.
CYR61 and CTGF are both immediate early genes, but the expression of r-CTGF was unchanged in response to mAChR stimulation in primary neurons. The CTGF promoter contains consensus sequences that are characteristic for growth factorinducible genes as well as a TGF-␤ response element, but different promoters have been postulated for CTGF in different tissues (34,64). Our data indicate that mAChR-related signaling do not regulate r-CTGF expression. NOV is expressed in quiescent cells and known to be down-regulated after serum stimulation in chicken embryo fibroblasts (36). Even though this appears in a PKC-dependent manner, we could not detect any down-regulation of NOVR after either 1 or 3 h of mAChR stimulation.
To determine whether mAChRs stimulate the expression of r-CYR61, r-CTGF, and NOVR in vivo, we analyzed forebrain regions of pilocarpine-and vehicle-treated rats and found that our data from primary neurons were confirmed by the in vivo situation. Whereas r-CYR61 was rarely detectable in unstimulated rat brain, the stimulation of mAChRs strongly increased r-CYR61 mRNA levels in cortical layers V, VI, and in thalamic nuclei. This special pattern is consistent with the innervation of the cortex by cholinergic neurons from the basal forebrain and with the known expression pattern of m1 and m3 mAChRs throughout the cortex, predominantly in cortical layer VI (2,3,65,66). Moreover, some thalamic nuclei are innervated by cholinergic neurons from the midbrain and nucleus basalis Meynert (2). In rat brain, mAChRs induce the expression of the immediate early genes c-fos, jun-B, and Egr-1/Krox24 in cortical layers IV, VI, and the hippocampus (53). Even though r-CYR61 revealed basal expression in hippocampus, the expression was not altered in response to mAChR stimulation in this brain region.
In contrast, r-CTGF message was predominantly present in the cortical layer VI, in both control and stimulated animals. Throughout the central nervous system, CTGF protein is found in astrocytes, as well as in neuronal cells of the cortex, where it supposedly mediates TGF-␤-related functions in cell growth, development, and tissue remodeling following injury (67,68). NOVR was expressed in cortical layers II/III, V, and VI, in the CA1-CA3 hippocampus region and the amygdala, which are major parts of the basal forebrain cholinergic system, but its expression did not change in response to mAChR stimulation. NOV is expressed during development of the central nervous system, and its protein is supposed to have important functions in the maintenance of differentiated neurons (40,69). Interestingly, we found r-CYR61, r-CTGF, and NOVR to be expressed in cells of cortical layer VI near corpus callosum. Kondo et al. (67) detected CTGF protein in rat brain in cortical layers III and V, and Su et al. (40) localized NOV mRNA in cortical layers V and VI of 38-week human embryos. Efferents and afferents of neurons in cortical layer VI project to other cortical layers. Therefore, differences between mRNA and protein localization especially of CTGF may be related to neuronal protein transport rather than diffusion of the secreted proteins. In addition to that afferents from pyramidal cells in cortical layer VI innervate the thalamus (70).
Our data suggest that CYR61 protein has a role in mediating mAChR-related alterations in synaptic plasticity. CYR61 encodes a secretory protein with functions in extracellular signaling; it binds to integrin ␣ v ␤ 3 and ␣ IIb ␤ 3 at the cell surface, as well as to heparin-containing components of the extracellular matrix (71)(72)(73). These interactions promote cell proliferation, adhesion, chemotaxis, and migration (37). If CYR61 has similar functions in brain cells, our studies suggest the possibility that it could be involved in the remodeling of neuronal connections, known to be associated with muscarinic receptor signaling. CYR61 may act, in concert with integrins, as a chemotactic factor that influences neurite outgrowth in response to a stimulating acetylcholine signal. This possibility is underscored by the known functions of brain integrins in neuronal differentiation, migration, neurite guidance, and long term potentiation (74 -77). CTGF has CYR61-related functions; in addition to that it regulates the expression of extracellular matrix components (34,38). Therefore, it may influence structural changes in neuronal plasticity in general, whereas NOV may support differentiated stages of neuronal systems.
Muscarinic signaling in cortex and hippocampus is impaired in Alzheimer's disease, whereas binding of ligands to mAChRs appears to be largely intact (4,5). This led to the development of cholinomimetic drugs and to the use of several acetylcholine esterase inhibitors for the therapy of Alzheimer's disease (78). Future studies are required to show whether h-CYR61 expression in brain is altered in response to the degeneration of subcortical projection neurons in Alzheimer's disease or other neurodegenerative diseases, and whether treatments with cholinomimetic drugs modulate its expression.