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Endocrinology, doi:10.1210/en.2007-0941
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Endocrinology Vol. 148, No. 12 5865-5873
Copyright © 2007 by The Endocrine Society

Human Chorionic Gonadotropin Induces Neuronal Differentiation of PC12 Cells through Activation of Stably Expressed Lutropin/Choriogonadotropin Receptor

Xing-Li Meng, Owen M. Rennert and Wai-Yee Chan

Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Wai-Yee Chan, Ph.D., Section on Developmental Genomics, Laboratory of Clinical Genomics, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 2A08, 49 Convent Drive, MSC 4429, Bethesda, Maryland 20892-4429. E-mail: chanwy{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human chorionic gonadotropin (hCG) and LH play an important role in reproductive physiology. Both hCG and LH bind to the same LH/choriogonadotropin receptor (LH/CG-R). Recent reports documented the temporal and spatial expression of LH/CG-R in the developing and mature mammalian brain. Administration of hCG promoted nerve regeneration in vivo and neurite outgrowth and survival of primary neurons in vitro. The function of hCG/LH and LH/CG-R in the nervous system remains unclear. In this study, we report that hCG/LH induced distinct morphological and biochemical changes, characteristic of neuronal differentiation, in PC12 cells stably expressing LH/CG-R and that the differentiation effect is ligand dose and time dependent. Western blot analysis revealed that both the ERKs and p38 MAPK are activated after hCG treatment. Inhibitor studies showed both the ERK and p38 MAPK signal transduction pathways are required for this differentiation process, which is cAMP dependent and protein kinase A independent. These findings imply a potential role for hCG/LH and LH/CG-R in the development, maintenance, and regeneration of the mammalian nervous system, and in the neuropathogenesis of genetic diseases caused by a mutated LH/CG-R.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN CHORIONIC gonadotropin (hCG) is a heterodimeric glycoprotein hormone that belongs to the cysteine-knot growth factor family (1). The syncytiotrophoblast of human placenta is the major site of hCG production. hCG is structurally and functionally similar to LH, which is primarily secreted by the anterior pituitary gland. Low levels of hCG or LH were also found in cancer tissues, cerebrospinal fluid, and central nervous system (CNS) tissues (2, 3, 4), suggesting possible local production of these hormones. Both hCG and LH bind to the same receptor, LH/choriogonadotropin receptor (LH/CG-R), which is a member of the G protein-coupled receptor (GPCR) family (5). hCG/LH and their receptor play an important role in reproductive physiology. In the ovary, hCG, through activation of LH/CG-R, is important for pregnancy maintenance by increasing the production of progesterone. In the testis, maturation of Leydig cells depends on testosterone production induced by hCG via activation of LH/CG-R; this is essential for male sexual development. During puberty, LH regulates steroidogenesis of Leydig cells through activation of LH/CG-R.

Nongonadal functions of hCG/LH were documented in recent years, and studies showed that LH/CG-R is expressed in a wide range of nongonadal tissues including those of the nervous system (6, 7). In rat embryonic brain, LH/CG-R protein levels are dynamically regulated (8). Its presence was detected from embryonic d 14.5, sharply increased thereafter, peaked at embryonic d 19, and gradually decreased after birth. In the adult mammalian CNS, LH/CG-R expression was found in brain regions such as the hippocampal formation, hypothalamus, cerebral cortex, brainstem, cerebellum, pituitary gland, neural retina, the ependymal region, and spinal cord (9, 10, 11, 12). Both neurons and glial cells were shown to express LH/CG-R (13, 14, 15). In culture systems, hCG has been reported to promote the survival and neurite outgrowth in primary neurons, possibly through activation of LH/CG-R (13). Animal studies demonstrated that injection of hCG improved recovery of motor function in rats with complete spinal cord transection (16, 17). These observations suggest that hCG/LH and LH/CG-R may play a role in the developing and mature nervous system.

It is well known that growth factors such as nerve growth factor (NGF) and neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) induce neuronal differentiation in vitro through activation of receptor tyrosine kinases (18, 19, 20) or GPCRs (21), respectively. It is intriguing that hCG belongs to the same cysteine-knot growth factor family as NGF and that LH/CG-R, like the receptor of PACAP, PAC1, is a member of the GPCRs that shares similar structural features with PAC1. We postulated that LH/CG-R and its ligands may participate in neuronal differentiation. The bipotential cell line, PC12, derived from rat adrenal pheochromocytoma, is an ideal in vitro model and has been extensively used for studying neuronal differentiation and signaling (18, 22). In this study, we show that hCG induced neuronal differentiation in PC12 cells ectopically expressing LH/CG-R mainly through the activation of ERKs and p38 MAPK signal transduction pathways. These results suggest a potential role of hCG/LH and their receptor in the neurogenesis of the mammalian nervous system and neural regeneration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
PC12 parental cells (American Type Culture Collection, Manassas, VA) and subclones were cultured in F-12K nutrient mixture with Kaighn’s modification (Invitrogen, Carlsbad, CA) containing 15% horse serum and 2.5% fetal bovine serum and 1% antibiotic and antimycotic on collagen type I-coated dishes (BD Biosciences, Bedford, MA) in a humidified incubator at 37 C with 5% CO2.

Plasmids and transfection
Human LH/CG-R cDNA with or without the single amino acid replacement of Asp578 by His (D578H) were cloned into pIRES2EGFP (Clontech, Mountain View, CA) to generate pLH/CG-R-IRESEGFP and pD578H-LH/CG-R-IRESEGFP. For transient expression study, cells were transfected using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instruction at 80% confluency. For stable transgene-expressing cell lines, cells were transfected with pLH/CG-R-IRESEGFP or empty vector, pIRES2EGFP, as described above. Seventy-two hours after transfection, cells were replated and selected with 0.4 mg/ml Geneticin (Invitrogen) for 2 wk. Drug-resistant clones expressing enhanced green fluorescent protein (EGFP) were isolated and expanded. The PC12/LHR and PC12/EGFP subclones were similar to their parental cells in aspects of morphology and growth rate.

RT-PCR detection of transduced (human) and endogenous (rat) LH/CG-R
Total RNA was isolated by RNeasy Mini Kit (QIAGEN, Valencia, CA) from cells transduced with vector and LH/CG-R with or without D578H mutation. RT was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with 2 µg total RNA following instructions of the manufacturer. Primers specific to human LH/CG-R (sense primer, 5'-ACATTTGCTAATCTCCTGGAG-3'; reverse primer, 5'-GCATCTGGTTCAGGAGCACA-3') were used to detect transduced LH/CG-R. The reaction was carried out at melting temperature 55 C for 35 cycles. The absence of DNA contamination was confirmed by reactions using RNA template without reverse transcriptase.

Endogenous rat LH/CG-R was detected in naive PC12 cells by nested RT-PCR as described previously (9) with modification. The following primers were used: for the first PCR, sense 5'-CTGGATATTTCTTCCAC-3' and reverse 5'-TGGCGTGGTTATAGTACTGGC-3', amplicon 612 bp; for the second PCR, sense 5'-AATTCACGAGCCTCCTGGTC-3' and reverse 5'-GCATCTGGTTCTGGAGCACA-3', amplicon 255 bp. Both reactions were carried out at melting temperature 55 C for 35 cycles. The amplified PCR product was verified by HhaI digestion and DNA sequencing.

Neurite outgrowth assay
Neurite outgrowth was examined at 72 h after transfection or addition of hCG or LH. For transfection experiments, cells with neurites longer than one times the diameter of the cell body were taken as differentiated cells. Cells with neurites between one and two times or more than two times the diameter of the cell body were counted separately. For hCG-stimulated PC12/LHR and PC12/EGFP cells, neurites longer than 1.5 times the diameter of the cell body were taken as differentiated cells. Cells from four randomly chosen optical fields were examined per dish, the numbers counted were pooled, and the percentage of differentiated cells was calculated based on the total number of cells appearing on the four fields regardless of neurite outgrowth. Total cell numbers analyzed for each group was approximately 1000–2000. Three dishes were analyzed for each group. The fold of induction of differentiation was calculated by the percentage of differentiated cells after treatments over that in untreated controls.

hCG and inhibitor treatment
Cells were serum starved overnight before hCG or LH treatment. hCG (C-0684, chorionic gonadotropin from human pregnancy urine; Sigma Chemical Co., St. Louis, MO) or LH (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD) in 10x diluted PC12 complete medium with F-12K was added to cells at different concentrations. In some experiments, 50 or 100 ng/ml 2.5S-NGF (murine, natural; Invitrogen) was used as controls. For inhibitor studies, cells were preincubated with inhibitors in 10x diluted PC12 complete medium at 37 C for 2 h (for U0126), 1 h (for U-73122 and U-73343), or 30 min (for SB203580, SP600125, LY294002, SQ22536, KT5720, and H-89), and then medium was replaced with fresh medium containing 400 ng/ml hCG and incubation continued for another 72 h. The inhibitors U0126 and LY294002 were obtained from Cell Signaling Technology (Beverly, MA). SB203580, SP600125, KT5720, U-73122, and U-73343 were from Sigma. H-89 was from EMD Biochemicals Inc (San Diego, CA).

Immunostaining
Cells were fixed with 2% paraformaldehyde in PBS at room temperature for 15 min and methanol at –20 C for 10 min. After blocking in 10% BSA in PBS with 0.3% Triton X-100 at room temperature for 2 h, cells were incubated with mouse monoclonal antibody to ß-tubulin III (1:200, clone TUJ-1; Fitzgerald, Concord, MA) or neurofilament 68 (NF68) (1:500; Sigma) in 5% BSA/PBS/Triton X-100 for 2 h at room temperature or overnight at 4 C, respectively. Cells were then reacted with rhodamine-conjugated goat antimouse IgG antibody. Signals were observed and photographed under an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany).

Western blot
After washing with PBS, cells were lysed in 1x RIPA buffer containing 25 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA), and 10 mM NaF. After centrifuging at 12,000 rpm for 5 min at 4 C, supernatants (about 6 µg total protein) were denatured in NuPAGE sample buffer (Invitrogen) and separated in 4–12% NuPAGE Bis-Tris gel (Invitrogen) and blotted to 0.2 µM polyvinylidene difluoride membrane (Invitrogen). The membranes were reacted with primary antibodies against phospho-p44/42, phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), phospho-p38 MAPK, phospho-Akt (Ser473), phospho-Akt (Thr308), total Akt, total p38, total SAPK/JNK (1:1000; Cell Signaling Technology), total p44/42 (1:2000; Upstate Cell Signaling Solutions, Lake Placid, NY), ß-tubulin III (1:1000, clone TUJ-1; Fitzgerald), and ß-actin (1:10,000; Sigma) in 5% BSA in PBS with 0.1% Tween 20 at 4 C overnight (except ß-tubulin III and ß-actin were incubated at room temperature for 1 h) and subsequently reacted with horseradish peroxidase-linked secondary antibodies at room temperature for 1 h. Signals were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL). Some membranes were stripped in Restore Western Blot Stripping Buffer (Pierce) at room temperature for 20 min for subsequently reacting with other primary antibodies.

Ras and Rap1 activation assay
Cells were grown at 80–90% confluency on 100-mm dishes and stimulated with hCG (1000 ng/ml) for 5 or 30 min. Ras or Rap1 activation assay kit (Upstate Cell Signaling Solutions) was used for the assay. Briefly, cells were lysed in 1x Mg lysis wash buffer or 1x Rap1 activation lysis buffer with 25 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail, and 10 mM NaF. Positive and negative controls were loaded either with GTP or GDP. Ras/Rap1 pull-down assays were performed by adding 10 µl Raf-1 ras binding domain agarose or 30 µl Ral GDS-rap binding domain agarose slurry and incubating at 4 C for 50 min. After washing three times with washing buffer, beads were resuspended with 20 µl NuPAGE sample buffer (Invitrogen) and separated in 12% NuPAGE Bis-Tris gel (Invitrogen) and blotted to a 0.2-µm polyvinylidene difluoride membrane (Invitrogen). The membranes were reacted with 1 µg/ml anti-Ras or anti-Rap1 antibody in 3% milk in PBS at 4 C overnight, and signal was detected as described above.

cAMP assay
The assay was performed with cAMP Biotrak enzyme immunoassay system (Amersham Biosciences, Piscataway, NJ) following the manufacturer’s manual. Cells were seeded in 35-mm dishes (for transient transduction) or 48-well plates (for stable clones). Three dishes or wells were analyzed for each treatment. Data are expressed as femtomoles per dish or well.

Statistical analysis
Student’s t test was used to compare cAMP content. {chi}2 analysis was used to compare percentages of differentiated cells. Mann-Whitney U test analysis was used to compare folds of induction of differentiation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of a constitutively activating LH/CG-R induces neuritogenesis of PC12 cells
hCG exerts its effect through the activation of its receptor, LH/CG-R. To determine whether direct LH/CG-R activation induces neuronal differentiation in PC12 cells, human LH/CG-R cDNA with an Asp578His mutation (D578H-LH/CG-R) was transiently expressed in PC12, and its effects were studied. The D578H mutation was originally found in Leydig cell adenomas (23). This mutant receptor was used as an analog to the agonist-activated wild-type (wt) receptor because it was shown to display constitutive activation toward both cAMP and inositol phosphate responses in Leydig cells, and the levels are close to those generated in LH/CG-R activation by high concentrations of hCG (24). Expression of the transduced human LH/CG-R with or without mutation was confirmed by 1) the presence of EGFP fluorescence (transfection efficiencies were similar in each transfection as examined by the percentages of EGFP-positives cells, about 15%), 2) RT-PCR using primers specific to human LH/CG-R (Fig. 1AGo, top), and 3) 72 h after transfection, cells transfected with D578H had significantly higher basal intracellular cAMP levels than those transfected with wt LH/CG-R or vector alone (P < 0.01) (Fig. 1AGo, bottom).


Figure 1
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  		FIG. 1. Overexpression of constitutively activating LH/CG-R induced PC12 neuritogenesis. PC12 cells were transfected with plasmids containing human LH/CG-R cDNA (wt), LH/CG-R carrying Asp578His mutation (D578H), or vector alone (vector). A, Top, The expression of transduced LH/CG-R was confirmed by RT-PCR; bottom, basal cAMP level in cells 72 h after transfection. *, P < 0.01, Student’s t test. Data are presented as means ± SE. B, Seventy-two hours after transfection, numbers of cells bearing long neurites (arrows) were significantly higher in D578H transfected PC12 cells than those transfected with wt or vector alone. C, Percentages of differentiated cells. *, P < 0.0001, {chi}2 test. Data are presented as means ± SE. D, Percentages of cells with different neurite lengths among differentiated cells.

 
After transduction with D578H-LH/CG-R, many PC12 cells extended neurites (Fig. 1BGo). The percentage of neurite-positive cells (more than one times the diameter of the cell body) was significantly higher than that in wt LH/CG-R and mock vector transfected PC12 cells (10.8 ± 1.8 vs. 3.4 ± 0.7 and 3.9 ± 0.8%, respectively, P < 0.0001) (Fig. 1CGo). Among neurite-positive cells, there were more cells with longer neurites (more than twice the diameter the cell body) in D578H-LH/CG-R transfected PC12 cells than those in wt LH/CG-R and vector transfected controls (Fig. 1DGo). No significant difference was observed in LH/CG-R and vector transfected cells. These results demonstrate direct activation of LH/CG-R induces neuritogenesis of PC12 cells.

hCG/LH-induced neuronal differentiation of PC12 cells
The effect of ligand-gated LH/CG-R activation on PC12 differentiation was further studied. Using nested RT-PCR, we detected endogenous rat LH/CG-R mRNA in PC12 cells only after extensive amplification (35 cycles of RT-PCR followed by 35 cycles of nested PCR), suggesting that the level of endogenous LH/CG-R was very low (Fig. 2AGo). To achieve sufficient LH/CG-R expression, wt human LH/CG-R was transduced into PC12 cells, and stable transgene-expressing subclones were established (PC12/LHR). hCG treatment led to significant elevation of intracellular cAMP in PC12/LHR cells but not in mock cells (PC12/EGFP), indicating the transduced LH/CG-R was functional (25). Treatment with hCG led to significant neuritogenesis of PC12/LHR cells when compared with untreated controls (P < 0.0001) (Fig. 2BGo). No significant increment in the number of neurite-positive cells was observed in PC12/EGFP cells after hCG treatment (P > 0.15), indicating that hCG alone did not lead to observable PC12 differentiation. Addition of LH also induced PC12/LHR neuritogenesis, although the percentages of neurite-positive cells were slightly lower than those with hCG at the same concentration (Fig. 2BGo). This may be due to the lower affinity of LH to LH/CG-R than hCG (26). In addition, to quantify the extent of PC12 differentiation by hCG, one clone each of PC12/EGFP (v1) and PC12/LHR (w1) were treated with 50 ng/ml NGF, and the fold induction of differentiated cells was compared with that of those treated with hCG (Fig. 2CGo).


Figure 2
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  		FIG. 2. Neuronal differentiation of PC12/LHR by hCG or LH. A, Naive PC12 cells express low levels of endogenous rat LH/CG-R confirmed by nested RT-PCR and PCR product was verified by enzyme digestion. B, Three subclones of PC12/LHR (w1, w3, and w5) and one PC12/EGFP clone (v1) were treated with 400 ng/ml hCG or LH. Percentages of differentiated cells were analyzed 72 h later. Data are presented as means ± SE. All PC12/LHR subclones showed significant increase of differentiated cells after hCG or LH treatment. *, P < 0.0001, {chi}2 test. No significant differences were observed in PC12/EGFP clones with or without hCG or LH treatment. **, P > 0.15, {chi}2 test. C, Fold induction of differentiated cells in clone v1 and w1 treated with 400 ng/ml hCG or 50 ng/ml NGF. D, Phase contrast microscopy and immunofluorescence of hCG-treated PC12/LHR cells (clone w5). Many hCG-treated cells had round, bright, and enlarged soma and multipolar neurites (arrow) and were positive for neuronal marker ß-tubulin III and NF68.

 
Neurite outgrowth in PC12/LHR cells became obvious morphologically at 24 h after hCG or LH treatment, and the neurites were often bipolar, similar to that reported for PACAP differentiated cells (27). At 72 h, the number of neurites increased and the neurites were very extended. Many cells had round, bright, and enlarged cell bodies with ramified neurites. (Fig. 2DGo, arrow) The morphology is similar to NGF differentiated cells, which often exhibit a neuronal-like perikaryon with branched neurites. At 5–6 d, numerous neurite connections were formed among the neurite-positive cells. The morphology of differentiated cells by LH was indistinguishable from hCG-treated cells. Many morphologically differentiated cells stained positively for the neuron-specific markers ß-tubulin III and NF68 (Fig. 2DGo). Activation of LH/CG-R by its ligands leads to neuronal differentiation of PC12 cells, and the differentiation effect of D378H-LH/CG-R on PC12 observed is not a phenomenon resulting from this specific mutation but rather an effect of the activation of LH/CG-R.

PC12 neuritogenesis was hCG dose and time dependent
The number of differentiated cells in hCG-treated PC12/LHR cells increased with increasing concentrations of hCG (100–1000 ng/ml) (Fig. 3Go, A and C). Neurite-positive cells could be clearly observed at 24 h, and the number of neurite-positive cells continued to increase at 48 and 72 h (Fig. 3Go, B and C). Consistent with these morphological observations, the ß-tubulin III expression level was also hCG dose and time dependent (Fig. 3DGo).


Figure 3
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  		FIG. 3. Neuritogenesis of PC12/LHR was hCG dose and time dependent. A, PC12/LHR (clone w5) cells were treated with various concentrations of hCG for 24 h. More neurite-positive cells could be observed in higher concentrations. B, The w5 cells were treated with 400 ng/ml hCG for 24, 48, and 72 h. More neurite-positive cells could be observed with longer incubation times. C, Quantitative assessment of neurite outgrowth in the conditions described in A and B. D, Western blot analysis showed hCG dose- and time-dependent induction of neuronal marker ß-tubulin III. ß-Actin was used as internal control. Insets in A and B, High-magnification view of representative cells.

 
Sustained activation of ERKs and p38 MAPK was required for hCG-induced PC12 differentiation
We investigated the intracellular signal transduction pathways involved in hCG-induced neuritogenesis in PC12/LHR cells. LH/CG-R activation by hCG led to increased phosphorylation of ERK1/2 at Thr202 and Tyr204 as early as 5 min. This phosphorylation diminished at 60 min but remained above the resting level up to 240 min (Fig. 4AGo). When cells were preincubated with the MAPK kinase-specific antagonist U0126, the neuritogenesis of PC12/LHR induced by hCG was inhibited. The inhibition was significant at 10 and 50 µM (P < 0.05) (Fig. 4BGo). Induction of NF68 expression by hCG was also inhibited by U0126 (Fig. 4CGo).


Figure 4
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  		FIG. 4. Activation of MAPK and inhibitor studies. A, Western blot showed changes in the phosphorylation states of MAPK in PC12/LHR (clone w5) cells treated with 1000 ng/ml hCG. Cells treated with NGF (100 ng/ml) for 5 min were used as control. B, Cells were preincubated with different concentrations of inhibitors of the ERK (U0126), p38 (SB203580), JNK (SP600125), and PI3K (LY294002) pathways followed by hCG (400 ng/ml) treatment for 72 h. Neurite outgrowth assay was performed. *, P < 0.05, Mann-Whitney U test. Data are presented as means ± SE. C, Western blot showing NF expression in inhibitor studies 72 h after addition of hCG.

 
In addition, hCG was observed to stimulate the phosphorylation of p38 MAPK at Thr180 and Tyr182 at both 5 and 240 min. No significant activation was observed at 60 and 120 min (Fig. 4AGo). Pretreating PC12/LHR cells with p38-specific inhibitor, SB203580, showed dose-dependent suppression of the hCG effect on the induction of neurite outgrowth (Fig. 4BGo). SB203580 also inhibited NF68 induction (Fig. 4CGo). These results suggest that activation of both ERK and p38 MAPK pathways are required for neuronal differentiation of PC12 induced by hCG.

The JNK and phosphoinositide 3-kinase (PI3K)/Akt pathway was not essential for PC12 differentiation
Phosphorylated SAPK/JNK gradually increased from 5 to 240 min after hCG treatment (Fig. 4AGo). Pretreatment with the inhibitor of JNK, SP600125, showed no inhibition but an increment of neurite-positive cells at 1 and 10 µM (Fig. 4BGo). This suggests JNK activity is not essential for neuronal differentiation of PC12/LHR cells induced by hCG.

There was a decrease in the phosphorylation of Akt both at Ser473 and Thr308 at 5 min up to 240 min compared with controls. (Fig. 4AGo) Because Akt is the downstream effector of PI3K, LY294002, the PI3K inhibitor, was used to study the involvement of Akt in hCG-induced neuritogenesis. Preincubating the cells with the inhibitor showed a slight increment of neurite-positive cells at 10 µM, which became significant at 50 µM (P < 0.05) (Fig. 4BGo). These results indicate inhibition of the JNK and Akt pathway does not prevent hCG-induced neuronal differentiation of PC12/LHR cells.

Role of cAMP in the neuronal differentiation of PC12/LHR cells
cAMP was previously shown to be an important component of the signaling network for PC12 differentiation (18, 28). Intracellular cAMP in PC12/LHR cells increased significantly after hCG treatment at concentrations from 100-1000 ng/ml. The higher the hCG concentration, the higher the level of cAMP detected (Fig. 5AGo). The level of cAMP started to increase at 1 min after hCG treatment (400 ng/ml), rapidly elevated up to 30 min, and dropped at 120 min. The inhibitor of adenylyl cyclase (AC), SQ22536, inhibited cAMP production and neurite outgrowth in PC12/LHR cells after hCG treatment. The inhibition was significant at 1 mM (P < 0.05) compared with non-inhibitor-treated controls (Fig. 5BGo). The requirement of protein kinase A (PKA), the cAMP-dependent protein kinase, for hCG-induced PC12 neuritogenesis was further examined. No inhibition of neurite outgrowth of PC12/LHR cells was observed when the PKA inhibitors KT5720 and H-89 were used (Fig. 5CGo). These results suggest that cAMP plays a crucial role in hCG-induced PC12 differentiation and that it is not PKA dependent.


Figure 5
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  		FIG. 5. Involvement of cAMP in hCG-induced PC12/LHR neuritogenesis. A, Intracellular cAMP assay: left, in PC12/LHR (clone w5) and PC12/EGFP (clone v1) cells at 30 min after treatment with different concentrations of hCG; right, in clone w5 for different lengths of time after treatment with 400 ng/ml hCG. B, AC inhibitor SQ22536 inhibited cAMP production (assay at 2 h after hCG treatment) and the hCG-induced neuritogenesis in PC12/LHR (clone w5) cells. *, P < 0.05, Mann-Whitney U test. C, Pretreatment with PKA inhibitors KT5720 and H-89 showed no effect on PC12/LHR neuritogenesis.

 
Phospholipase C (PLC) pathway is partially involved in hCG-induced PC12 differentiation
We investigated the possible involvement of the PLC pathway in hCG-induced PC12 neuritogenesis. The PLC inhibitor U-73122 at 5 µM of concentration inhibited neurite outgrowth from 22.4–11.5%, whereas its inactive analog U-73343 did not inhibit neurite outgrowth (Fig. 6AGo). The inhibition by U-73122 was partial, because the number of differentiated cells was still much higher in U-73122-treated cells than untreated controls (11.46 vs. 2.94%). Because significant cell death was observed when higher doses (7.5 and 10 µM) of U-73122 and U-73343 were used, it is not likely that the partial inhibition was due to an insufficient concentration of inhibitor. We also investigated the enhancement of Rap1 and Ras GTP induced by hCG. No Ras activation was observed after hCG stimulation. However, there was slight elevation of Rap1 GTP loading at 30 min after hCG stimulation (Fig. 6BGo).


Figure 6
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  		FIG. 6. Involvement of PLC pathway and Rap1 in hCG-induced neuritogenesis. A, PC12/LHR (clone w5) cells were preincubated with various concentrations of the PLC inhibitor U-73122 or its inactive analog U-73343 followed by hCG (400 ng/ml) treatment for 72 h. Neurite outgrowth assay was performed. *, P < 0.0001, {chi}2 test. Data are presented as mean ± SE. B, PC12/LHR (clone w3) cells were treated with hCG for 5 and 30 min. GTP loading of Ras and Rap1 was analyzed by immunoprecipitation and Western blotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated that hCG/LH induced neuronal differentiation of PC12 cells through LH/CG-R activation. hCG induced morphological changes characteristic of neuronal differentiation including neurite outgrowth, neuron-like enlarged soma, formation of neurite network, and expression of neuronal markers. The differentiation process is primarily mediated by activation of ERK and p38 MAPK signal transduction pathways.

The concentrations of hCG used in this study were from 200-1000 ng/ml. These high concentrations were chosen based on previous studies (13, 15), in which hCG effects on primary neurons and glial cells were significant only from 100–250 ng/ml. These studies did not provide evidence that the hCG effects were through LH/CG-R activation; our study documents clear evidence for the requirement of LH/CG-R activation to produce neuronal differentiation by hCG treatment in PC12 cells. First, in the absence of ligand, transduction of an activating mutant receptor, but not the wt receptor, led to neuronal differentiation of PC12 cells. Second, both ligands, hCG and LH, induced differentiation of PC12/LHR cells but not in mock controls (PC12/EGFP). Last, hCG induced cAMP production in PC12/LHR cells but not in mock controls, and inhibition of cAMP production inhibited hCG-induced neuritogenesis.

Although we documented endogenous LH/CG-R mRNA expression in naive PC12 cells by RT-PCR, no differentiation was observed in cells transduced with empty vector (PC12/EGFP) after hCG treatment (Fig. 2BGo). Accordingly, cAMP production after hCG treatment was minimal in these cells (Fig. 5AGo). These data suggest that the expression level of endogenous LH/CG-R may be too low and its activation may not be sufficient to show the differentiation effect. An alternate explanation for the unresponsiveness is that the endogenous receptor may be functionally impaired. These results provide new insights into the role of hCG/LH and their receptor in the nervous system and potentially have important implications in the development, maintenance, and regeneration of the CNS as well as the neuropathogenesis of genetic diseases caused by mutated LH/CG-R. Based on the dynamically regulated expression of LH/CG-R in rat embryonic brain, and the high concentration of hCG present in maternal and fetal blood streams, especially during the first trimester of gestation in humans (29), it is highly possible that hCG-induced LH/CG-R activation may participate in neuronal differentiation and maturation of the developing CNS. In the adult mammalian brain, although the majority of neurons are postmitotic, there are multipotent neural stem/progenitor cells generating young neurons throughout life (30, 31). The expression of LH/CG-R in the dentate gyrus of the hippocampal formation and the ependymal region, major sites known to harbor progenitor cells (32), suggests the possibility that LH-induced LH/CG-R activation may be involved in neuronal renewal in the mature CNS. In animal models of spinal cord injury, administration of hCG improved neuronal regeneration. This phenomenon could be explained, at least in part, by neuronal generation from progenitor cells through LH/CG-R activation. These observations suggest it might be useful to study the potential role of LH/CG-R up-regulation at such injury sites. The differentiation effect of LH/CG-R activation shown in the present study provides an alternate mechanism, in addition to the elevated sex hormone testosterone, for behavioral abnormalities observed in familial male-limited precocious puberty patients who harbor activating mutations in LH/CG-R (25, 33). Because differentiation during development is a stringently controlled process, it is possible that aberrant continuous activation of LH/CG-R may affect the number and/or proportions of certain subtypes of neurons generated and thereby cause structural and/or functional abnormalities in the brain.

The documented hCG role in neuronal differentiation may have therapeutic significance for recovery from acute nerve injury as well as in neurodegenerative disorders. NGF, brain-derived neurotrophic factor, and other neurotrophic factors have been used in the treatment of chronic neurodegenerative disorders and spinal cord injury by promoting neuronal differentiation, survival, and synaptic plasticity (34). hCG may be an alternative candidate for the treatment of these neuropathological conditions. Because hCG can cross the blood-brain barrier, it would make drug delivery easier. Because hCG is present during pregnancy at high levels with no severe side effects, its pharmacological availability and safety may be another advantage (35). Future investigations are needed to explore the possible therapeutic application of this hormone.

Neuronal differentiation induced by hCG needs the integration of signals from multiple transduction pathways. We documented that sustained activation of both ERKs and p38 MAPK is indispensable for the differentiation effect by inhibitor studies. The role of ERK pathway in PC12 differentiation has been extensively studied (22, 28, 36). Recent studies showed that sustained ERK activation is required but not sufficient for growth factor-mediated PC12 differentiation (37). An essential role of P38 MAPK in PC12 differentiation has also been suggested (38). Other reports showed that in the absence of ERK activation, activation of the p38 pathway led to neuronal differentiation of PC12 cells (39). In our study, we demonstrated a requirement for both ERK and p38. This is consistent with the notion that a cellular outcome such as differentiation depends on the ligand-dependent generation of signaling pathways, strength of the pathways, and the unique combination of these pathways in a given cell type. Phosphorylation of p38 MAPK at Thr180 and Tyr182 were detected at both 5 and 240 min but not at 60 and 120 min. The reason for these cyclic changes in the phosphorylation state of p38 MAPK is unknown. Activation of the JNK and PI3/Akt pathways appears less important in the differentiation process examined. This is not surprising because JNK activity was proposed to be essential for the functional differentiation of a PC12 variant at a late stage of neuritogenesis, whereas the PI3/Akt pathway was mainly involved in cell survival (40, 41).

Because LH/CG-R and the receptor for PACAP are both members of GPCRs, hCG and PACAP may share similar mechanisms for signal generation after receptor activation. LH/CG-R has a long N-terminal extracellular domain, seven transmembrane helices and connecting loops, and a short C-terminal intracellular domain. The extracellular domain is responsible for ligand binding; the transmembrane and intracellular domains are important for signal transduction. It independently activates two G protein-dependent pathways, AC and PLC (42). It is possible that hCG, by activating LH/CG-R, induced both AC and PLC pathways to interact with the MAPK networks. In this study, we demonstrated that AC activity is essential for cAMP production and LH/CG-R activation-induced neuritogenesis, which implies that the neurite outgrowth effect is cAMP dependent, yet this process is apparently not PKA dependent. PKA activity is required for the sustained activation of ERK in NGF-stimulated PC12 neuritogenesis, whereas in studies with PACAP, the minimal contribution of PKA to PC12 neuritogenesis was reported, and activation of ERK kinase activity was primarily protein kinase C and MAPK kinase dependent (43). A recent report also suggested hCG uses a cAMP-dependent but PKA-independent signaling mechanism to up-regulate human placental indoleamine 2,3-dioxygenase (44). Because in PC12 cells protein kinase C activation is linked to PLC phospholipid hydrolytic activity, the PLC pathway may play an important part in the activation of ERKs induced by hCG. The PLC inhibitor U-73122 partially inhibited hCG-induced neuritogenesis, suggesting an involvement of the PLC pathway in the differentiation process. To study the potential link between cAMP, PLC, and MAPK activation, we assessed the activation of the small GTP-binding proteins Ras and Rap1 in hCG-stimulated PC12/LHR cells. There was no Ras activity detected, whereas there was a slight increment of GTP loading of Rap1. In PACAP-stimulated PC12 cells, ERK activation was not Ras dependent but rather through PLC-dependent Rap1 activation (45). In our study, Rap1 could also be a candidate for the ERK activation induced by hCG (46). Because Rap1 activation by hCG was not significant, it is not clear whether it is sufficient to induce ERK activation and whether other upstream components are involved in the early phase of ERK activation. In addition, whether Rap1 activation induced by hCG is PLC dependent or is a direct effect of cAMP (47) needs further study. Investigation of the signal transduction pathways involved in hCG-induced neuronal differentiation will enhance our understanding of the molecular mechanisms of LH/CG-R activation and the differentiation process.


   Footnotes
 
This research was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

Disclosure Summary: The authors have nothing to disclose.

First Published Online August 30, 2007

Abbreviations: AC, Adenylyl cyclase; CG-R, choriogonadotropin receptor; CNS, central nervous system; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; hCG, human chorionic gonadotropin; JNK, c-Jun N-terminal kinase; NF68, neurofilament 68; NGF, nerve growth factor; PACAP, pituitary adenylate cyclase-activating polypeptide; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PLC, phospholipase C; SAPK, stress-activated protein kinase; wt, wild type.

Received July 11, 2007.

Accepted for publication August 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW 1994 Crystal structure of human chorionic gonadotropin. Nature 369:455–461[CrossRef][Medline]
  2. Bagshawe KD, Orr AH, Rushworth AG 1968 Relationship between concentrations of human chorionic gonadotrophin in plasma and cerebrospinal fluid. Nature 217:950–951[CrossRef][Medline]
  3. Hostetter G, Gallo RV, Brownfield MS 1981 Presence of immunoreactive luteinizing hormone in the rat forebrain. Neuroendocrinology 33:241–245[Medline]
  4. Birken S, Maydelman Y, Gawinowicz MA, Pound A, Liu Y, Hartree AS 1996 Isolation and characterization of human pituitary chorionic gonadotropin. Endocrinology 137:1402–1411[Abstract]
  5. McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494–499[Abstract/Free Full Text]
  6. Rao CV 2001 An overview of the past, present, and future of nongonadal LH/hCG actions in reproductive biology and medicine. Semin Reprod Med 19:7–17[CrossRef][Medline]
  7. Lei ZM, Rao CV 2001 Neural actions of luteinizing hormone and human chorionic gonadotropin. Semin Reprod Med 19:103–109[CrossRef][Medline]
  8. AL-Hader AA, Tao YX, Lei ZM, Rao CV 1997 Fetal rat brains contain luteinizing hormone/human chorionic gonadotropin receptors. Early Pregnancy 3:323–329[Medline]
  9. Lei ZM, Rao CV, Kornyei JL, Licht P, Hiatt ES 1993 Novel expression of human chorionic gonadotropin/luteinizing hormone receptor gene in brain. Endocrinology 132:2262–2270[Abstract/Free Full Text]
  10. Thompson DA, Othman MI, Lei Z, Li X, Huang ZH, Eadie DM, Rao CV 1998 Localization of receptors for luteinizing hormone/chorionic gonadotropin in neural retina. Life Sci 63:1057–1064[CrossRef][Medline]
  11. Bukovsky A, Indrapichate K, Fujiwara H, Cekanova M, Ayala ME, Dominguez R, Caudle MR, Wimalsena J, Elder RF, Copas P, Foster JS, Fernando RI, Henley DC, Upadhyaya NB 2003 Multiple luteinizing hormone receptor (LHR) protein variants, interspecies reactivity of anti-LHR mAb clone 3B5, subcellular localization of LHR in human placenta, pelvic floor and brain, and possible role for LHR in the development of abnormal pregnancy, pelvic floor disorders and Alzheimer’s disease. Reprod Biol Endocrinol 1:46[CrossRef][Medline]
  12. Rao SC, Li X, Rao ChV, Magnuson DS 2003 Human chorionic gonadotropin/luteinizing hormone receptor expression in the adult rat spinal cord. Neurosci Lett 336:135–138[CrossRef][Medline]
  13. AL-Hader AA, Lei ZM, Rao CV 1997 Neurons from fetal rat brains contain functional luteinizing hormone/chorionic gonadotropin receptors. Biol Reprod 56:1071–1076[Abstract]
  14. Zhang W, Lei ZM, Rao CV 1999 Immortalized hippocampal cells contain functional luteinizing hormone/human chorionic gonadotropin receptors. Life Sci 65:2083–2098[CrossRef][Medline]
  15. AL-Hader AA, Lei ZM, Rao CV 1997 Novel expression of functional luteinizing hormone/chorionic gonadotropin receptors in cultured glial cells from neonatal rat brains. Biol Reprod 56:501–507[Abstract]
  16. Patil AA, Nagaraj MP 1983 The effect of human chorionic gonadotropin (HCG) on functional recovery of spinal cord sectioned rats. Acta Neurochir (Wien) 69:205–218[CrossRef][Medline]
  17. Patil AA, Filmore K, Hill D 1990 The effect of human chorionic gonadotropin (HCG) on restoration of physiological continuity of the spinal cord. A preliminary report. Int Surg 75:54–57[Medline]
  18. Greene LA, Tischler AS 1976 Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424–2428[Abstract/Free Full Text]
  19. Rydel RE, Greene LA 1987 Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci 7:3639–3653[Abstract]
  20. Heasley LE, Johnson GL 1992 The ß-PDGF receptor induces neuronal differentiation of PC12 cells. Mol Biol Cell 3:545–553[Abstract]
  21. Deutsch PJ, Sun Y 1992 The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J Biol Chem 267:5108–5113[Abstract/Free Full Text]
  22. Vaudry D, Stork PJ, Lazarovici P, Eiden LE 2002 Signaling pathways for PC12 cell differentiation: making the right connections. Science 296:1648–1649[Abstract/Free Full Text]
  23. Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA, Shenker A 1999 Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. N Engl J Med 341:1731–1736[Free Full Text]
  24. Hirakawa T, Galet C, Ascoli M 2002 MA-10 cells transfected with the human lutropin/choriogonadotropin receptor (hLHR): a novel experimental paradigm to study the functional properties of the hLHR. Endocrinology 143:1026–1035[Abstract/Free Full Text]
  25. Shenker A, Laue L, Kosugi S, Merendino JJ Jr., Minegishi T, Cutler Jr GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
  26. Bernard MP, Myers RV, Moyle WR 1998 Lutropins appear to contact two independent sites in the extracellular domain of their receptors. Biochem J 335:611–617[Medline]
  27. Grumolato L, Alexandre D, Turquier V, Ait-Ali D, Fournier A, Vaudry H, Anouar Y 2002 Pituitary adenylate cyclase-activating polypeptide regulates neuroendocrine markers and transcription factors in differentiating pheochromocytoma cells. Ann NY Acad Sci 971:467–470[Medline]
  28. Ravni A, Bourgault S, Lebon A, Chan P, Galas L, Fournier A, Vaudry H, Gonzalez B, Eiden LE, Vaudry D 2006 The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways. J Neurochem 98:321–329[CrossRef][Medline]
  29. Fisher DA 1998 Endocrinology of fetal development. In: Wilson JD, ed. Williams textbook of endocrinology. 9th ed. Los Angeles: University of California; 1273–1301
  30. Colucci-D’Amato L, Bonavita V, di Porzio U 2006 The end of the central dogma of neurobiology: stem cells and neurogenesis in adult CNS. Neurol Sci 27:266–270[CrossRef][Medline]
  31. Gage FH 2000 Mammalian neural stem cells. Science 287:1433–1438[Abstract/Free Full Text]
  32. Gage FH 2002 Neurogenesis in the adult brain. J Neurosci 22:612–613[Free Full Text]
  33. Weissenberger AA, Leschek EW, Zametkin AJ 2001 Case study: sexual hyperactivity treated with psychostimulants in familial male precocious puberty. J Am Acad Child Adolesc Psychiatry 40:373–376[CrossRef][Medline]
  34. Dawbarn D, Allen SJ 2003 Neurotrophins and neurodegeneration. Neuropathol Appl Neurobiol 29:211–230[CrossRef][Medline]
  35. Chao MV, Rajagopal R, Lee FS 2006 Neurotrophin signalling in health and disease. Clin Sci (Lond) 110:167–173[Medline]
  36. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185[CrossRef][Medline]
  37. Vaillancourt RR, Heasley LE, Zamarripa J, Storey B, Valius M, Kazlauskas A, Johnson GL 1995 Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation. Mol Cell Biol 15:3644–3653[Abstract]
  38. Morooka T, Nishida E 1998 Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells. J Biol Chem 273:24285–24288[Abstract/Free Full Text]
  39. Iwasaki S, Iguchi M, Watanabe K, Hoshino R, Tsujimoto M, Kohno M 1999 Specific activation of the p38 mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein-2. J Biol Chem 274:26503–26510[Abstract/Free Full Text]
  40. Xiao J, Liu Y 2003 Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system. J Neurochem 86:1516–1523[CrossRef][Medline]
  41. Ashcroft M, Stephens RM, Hallberg B, Downward J, Kaplan DR 1999 The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth. Oncogene 18:4586–4597[CrossRef][Medline]
  42. Gudermann T, Birnbaumer M, Birnbaumer L 1992 Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J Biol Chem 267:4479–4488[Abstract/Free Full Text]
  43. Lazarovici P, Jiang H, Fink Jr D 1998 The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase. Mol Pharmacol 54:547–558[Abstract/Free Full Text]
  44. Lei ZM, Yang M, Li X, Takikawa O, Rao CV 2007 Upregulation of placental indoleamine 2,3-dioxygenase by human chorionic gonadotropin. Biol Reprod 76:639–644[Abstract/Free Full Text]
  45. Bouschet T, Perez V, Fernandez C, Bockaert J, Eychene A, Journot L 2003 Stimulation of the ERK pathway by GTP-loaded Rap1 requires the concomitant activation of Ras, protein kinase C, and protein kinase A in neuronal cells. J Biol Chem 278:4778–4785[Abstract/Free Full Text]
  46. York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ 1998 Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. Nature 392:622–626[CrossRef][Medline]
  47. Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ 1997 cAMP activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent pathway. Cell 89:73–82[CrossRef][Medline]



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