| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 17, 2001 as doi:10.1096/fj.00-0867fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INSERM U 531, IFR 31, CHU Rangueil, 31403 Toulouse Cedex 4, France
3Correspondence: INSERM U 531, IFR 31, CHU Rangueil, 31403 Toulouse Cedex 4, France. E-mail: susinich{at}rangueil.inserm.fr
SPECIFIC AIMS
The sst2 somatostatin receptor is an inhibitory G-protein-coupled receptor that inhibits normal and tumor cell growth by a mechanism involving activation of the tyrosine phosphatase SHP-1. We addressed whether nitric oxide synthase (NOS) is involved in sst2 signaling. We first investigated whether an NOS isoform interacts with SHP-1 and is activated by somatostatin. We further studied NOS role for sst2-mediated negative growth signal regulation and identified the associated molecular mechanism.
PRINCIPAL FINDINGS
1. SHP-1 associates with neuronal NOS (nNOS) and is involved in
somatostatin-induced dephosphorylation of nNOS
To investigate whether SHP-1 interacts with NOS, we performed
coimmunoprecipitation and immunoblotting studies in CHO cells
expressing sst2 (CHO/sst2). nNOS, but not endothelial NOS, was detected
within anti-SHP-1 immunoprecipitates in resting cells. SHP-1 was also
coprecipitated within anti-nNOS immunoprecipitates. CHO/sst2 cell
treatment with RC-160 (1 nM), a sst2 agonist, increased the amount of
immunoprecipitated SHP-1/nNOS complexes, with a maximum at 1–3 min of
RC-160 treatment. Because RC-160 induced SHP-1 activity in CHO/sst2,
the demonstration of nNOS/SHP-1 interaction suggested that nNOS is a
substrate for RC-160-activated SHP-1. nNOS was tyrosine phosphorylated
in resting cells and cell treatment with RC-160 reduced nNOS tyrosine
phosphorylation. This effect was evident within 1 min of RC-160
treatment and reached a maximum at 5 min (Fig. 1A
). To test whether active SHP-1 is critical for
somatostatin-mediated decrease of nNOS tyrosine phosphorylation, we
used CHO cells stably expressing sst2 and a dominant-negative form of
SHP-1, referred to CHO/sst2-SHP-1(C453S) cells. In
CHO/sst2-SHP-1(C453S) cells, nNOS was hyperphosphorylated at the basal
level (Fig. 1C
) and RC-160 was unable to dephosphorylate
nNOS (Fig. 1B
). This result is consistent with a critical
role for active SHP-1 to mediate RC-160-induced nNOS tyrosine
dephosphorylation.
|
2. SHP-1 is required for somatostatin-induced activation of nNOS in
vitro
To determine whether SHP-1-mediated dephosphorylation of nNOS is
relevant to nNOS activation, CHO/sst2 cells were incubated in the
presence or absence of somatostatin agonist and nNOS activity was
examined in nNOS immunoprecipitates. CHO/sst2 cell treatment with
RC-160 induced nNOS activity, which reached 172 ± 13% at 5 min.
RC-160 had no effect on nNOS activity in CHO/sst2-SHP-1(C453S) cells.
Our results indicate that RC-160-activated SHP-1 dephosphorylates nNOS,
thereby increasing its activity.
3. SHP-1 is required for somatostatin-induced activation of nNOS in
vivo
To ascertain whether the regulation of nNOS phosphorylation and
activation by SHP-1 are physiologically relevant in vivo, we used
pancreatic acini isolated from normal mice that highly express
endogenous sst2 as well as from motheaten viable
(mev) mice carrying mutations in the
SHP-1 gene responsible for loss of SHP-1 activity. As observed in
CHO/sst2 cells, in acini from normal mice, nNOS was tyrosine
phosphorylated at the basal level and treatment with RC-160 resulted in
a decrease of nNOS tyrosine phosphorylation, associated with an
increase of nNOS activity. By contrast, in acini derived from
mev mice, RC-160 affected neither nNOS
tyrosine phosphorylation nor its catalytic activity. These results
provide direct evidence for in vivo RC-160-mediated nNOS
dephosphorylation and activation, and confirm the critical role of
SHP-1 for RC-160-induced nNOS activation.
4. Somatostatin-mediated activation of nNOS stimulates
intracellular cGMP production
Under physiological conditions, NO effects are partially mediated
by the stimulation of soluble guanylate cyclase, which produces
intracellular cGMP. Therefore, we next examined the effect of RC-160 on
the intracellular cGMP level. Stimulation of CHO/sst2 cells or normal
mouse acini with RC-160 for 5 min increased cGMP production. This
RC-160 effect was abrogated in CHO/sst2-SHP-1(C453S) cells and in acini
from mev mice, whereas the NO donor sodium
nitroprusside (SNP) still enhanced cGMP production in these cells.
These results indicate that blockade of RC-160 induced NO release and
that cGMP production in cells expressing an inactive SHP-1 was likely
due to an absence of nNOS stimulation.
5. Somatostatin-activated nNOS/NO/cGMP pathway is involved in
sst2-mediated antiproliferative signal
To obtain direct evidence for nNOS implication in RC-160-mediated
inhibition of cell proliferation, we transiently coexpressed sst2 and
the rat nNOS(C415A) mutant in CHO cells. The
Cys415 of nNOS(C415A) was mutated to Ala to
prevent binding of the heme and tetrahydrobiopterin cofactors that are
critical for nNOS activation; 1 nM RC-160 treatment for 3 min promoted
nNOS activation in CHO/sst2-pCMV5 but not in CHO/sst2-nNOS(C415A)
cells. In addition, expression of the catalytically inactive nNOS
abrogated the somatostatin analog inhibitory effect on cell
proliferation. RC-160 indeed inhibited FCS-induced CHO/sst2-pCMV5 cell
proliferation whereas proliferation of CHO/sst2-nNOS(C415A) cells
remained unaffected. Finally, expression of a dominant-negative form of
nNOS prevented RC-160-induced p27 up-regulation observed on CHO/sst2
cells, indicating that nNOS is involved and contributes to
sst2-mediated G1 cell cycle arrest.
We tested a synthetic inhibitors of both and both soluble guanylate cyclase (L-NAME and LY 83583, respectively) on the antiproliferative effect of RC-160. These inhibitors both significantly reduced RC-160-induced inhibition of CHO/sst2 cell proliferation In contrast, RC-160 did not modify CHO/sst2-SHP-1(C453S) cell proliferation, and the addition of LY 83583 or L-NAME had no effect on these cells. Moreover, the NO donor SNP and the cGMP analog 8-Br-cGMP inhibited serum-induced proliferation of both CHO/sst2 and CHO/sst2-SHP-1(C453S) cells. These results not only indicate that the NO/cGMP pathway is functional in both cell types, but also that this pathway is involved downstream of SHP-1 in the RC-160-induced negative growth signal. All together, our data argue in favor of a critical role for the SHP-1/nNOS/NO/cGMP pathway in sst2-initiated negative growth signal.
CONCLUSION
We previously reported that the inhibitory receptor sst2 mediated the antiproliferative effect of somatostatin by stimulating SHP-1 tyrosine phosphatase activity. Here we provide evidence for a direct regulation of nNOS by SHP-1 and demonstrate the role of nNOS in the sst2 growth inhibitory signal transduction pathway.
SHP-1 has been identified as a critical negative regulator of several
growth factor, antigen, and cytokine receptor signaling. SHP-1
recruitment to activated receptors or associated proteins indeed
results in inhibition of their signaling cascades. In hematopoietic
cells, it is well established that inhibitory immunoreceptors
(including killer inhibitory receptors) down-regulate signaling by
recruiting SHP-1. SHP-1 then dephosphorylates activated receptors and
their associated molecules that are involved in early signal
transduction and thereby terminates activating signals. Little is known
about the signaling effectors with which SHP-1 interacts to transduce
inhibitory G-protein-coupled receptor signals. We previously reported
that ligand-activated sst2 recruits SHP-1, which results in SHP-1
enzymatic activation and subsequently dephosphorylation of SHP-1
substrates. In this study, we have identified nNOS as a novel substrate
for sst2-activated SHP-1 in vitro in CHO cells expressing sst2, and
further demonstrated in vivo in the well-characterized sst2-expressing
mouse pancreatic acini a functional role of SHP-1 for nNOS regulation
after sst2 activation. The observed SHP-1-dependent effects of
somatostatin on tyrosine dephosphorylation and activation of nNOS are
consistent with results obtained both in vitro in CHO/sst2 cells
expressing a catalytically inactive SHP-1 and in vivo in mouse
pancreatic acini derived from mev mice
carrying inactivating mutations in the SHP-1 gene. In these cells, nNOS
can no longer be tyrosine dephosphorylated and activated in response to
somatostatin. Our results demonstrate the importance of SHP-1 for
regulating nNOS activity in the sst2-initiated signaling pathway. nNOS
enzyme activity is modulated by phosphorylation/dephosphorylation of
serine and threonine residues. However, emerging evidence suggests that
phosphorylation/dephosphorylation on tyrosine residues influence nNOS
enzyme activity. Indeed, lipopolysaccharide plus interferon 
increase nNOS tyrosine phosphorylation, which is associated with
inhibition of nNOS enzyme activity. Conversely, cholecystokinin-induced
activation of the tyrosine phosphatase SHP-2 results in tyrosine nNOS
dephosphorylation and activation. Taken together, these results argue
in favor of the role of specific tyrosine kinases and tyrosine
phosphatases in control of both the nNOS tyrosine phosphorylation level
and enzymatic activity. nNOS tyrosyl residues and mechanisms involved
in such a regulation remain to be identified.
The physiological role for somatostatin-mediated activation of nNOS in pancreatic acini is not yet established. However, because somatostatin was reported to inhibit pancreatic acinar cell growth, it can be suggested that nNOS contributes to pancreatic cell development.
Somatostatin stimulates cGMP production in CHO/sst2 cells and normal mouse pancreatic acini. Somatostatin-induced guanylate cyclase activation, however, was abrogated in cells expressing an inactive SHP-1, whereas the response to the exogenous NO donor SNP was not modified in these cells. These results suggest that SHP-1 is critical for sst2-mediated cGMP production.
The antiproliferative effect of somatostatin on CHO cells is blocked by
coexpressing sst2 and a catalytically inactive nNOS or by treating
these cells with the NOS competitive inhibitor, L-NAME or the specific
guanylate cyclase inhibitor LY 83583. SNP and 8-bromo-cGMP
mimic somatostatin’s effect on cell proliferation. Our results provide
evidence that the nNOS/NO/cGMP signaling pathway is involved in
sst2-mediated inhibitory growth signaling (Fig. 2
). The downstream effectors of sst2-induced cGMP production remain to be
elucidated.
|
There is widespread distribution of sst2 in the central and peripheral nervous systems as well as in peripheral organs. Sst2 is also highly expressed in the majority of human tumors. Sst2 is implicated in the regulation of various functions including inhibition of hormone secretions, cell proliferation and gut motility, and neurotransmission and behavior, and pathological situations such as tumor growth and inflammation. nNOS has been identified in different tissues including brain, skeletal muscle, gut, and pancreas, where sst2 expression occurs. It would be therefore of critical interest to investigate whether sst2 and nNOS colocalize and whether nNOS transduces other sst2 cellular functions in addition to its antiproliferative effect. The present findings that nNOS acts as an important molecule in the sst2-mediated growth inhibitory signaling represent a first step in demonstrating that nNOS participates in sst2-initiated cellular responses.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0867fje; to cite this article, use FASEB J. (August 17, 2001) 10.1096/fj.00-0867fje 
2 These two authors contribute equally to this work.
This article has been cited by other articles:
|
|
 |
|
  S. Arena, A. Pattarozzi, A. Massa, J.-P. Esteve, R. Iuliano, A. Fusco, C. Susini, and T. Florio An Intracellular Multi-Effector Complex Mediates Somatostatin Receptor 1 Activation of Phospho-Tyrosine Phosphatase {eta} Mol. Endocrinol., January 1, 2007; 21(1): 229 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cordelier, J.-P. Esteve, S. Najib, L. Moroder, N. Vaysse, L. Pradayrol, C. Susini, and L. Buscail Regulation of Neuronal Nitric-oxide Synthase Activity by Somatostatin Analogs following SST5 Somatostatin Receptor Activation J. Biol. Chem., July 14, 2006; 281(28): 19156 - 19171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Arena, A. Pattarozzi, A. Corsaro, G. Schettini, and T. Florio Somatostatin Receptor Subtype-Dependent Regulation of Nitric Oxide Release: Involvement of Different Intracellular Pathways Mol. Endocrinol., January 1, 2005; 19(1): 255 - 267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Lahlou, N. Saint-Laurent, J.-P. Esteve, A. Eychene, L. Pradayrol, S. Pyronnet, and C. Susini sst2 Somatostatin Receptor Inhibits Cell Proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 Activation J. Biol. Chem., October 10, 2003; 278(41): 39356 - 39371. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Thomas, M. R. Hellmich, C. M. Townsend Jr., and B. M. Evers Role of Gastrointestinal Hormones in the Proliferation of Normal and Neoplastic Tissues Endocr. Rev., October 1, 2003; 24(5): 571 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hortala, G. Ferjoux, A. Estival, C. Bertrand, S. Schulz, L. Pradayrol, C. Susini, and F. Clemente Inhibitory Role of the Somatostatin Receptor SST2 on the Intracrine-regulated Cell Proliferation Induced by the 210-Amino Acid Fibroblast Growth Factor-2 Isoform: IMPLICATION OF JAK2 J. Biol. Chem., May 30, 2003; 278(23): 20574 - 20581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Florio, M. Morini, V. Villa, S. Arena, A. Corsaro, S. Thellung, M. D. Culler, U. Pfeffer, D. M. Noonan, G. Schettini, et al. Somatostatin Inhibits Tumor Angiogenesis and Growth via Somatostatin Receptor-3-Mediated Regulation of Endothelial Nitric Oxide Synthase and Mitogen-Activated Protein Kinase Activities Endocrinology, April 1, 2003; 144(4): 1574 - 1584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Fleming and R. Busse Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2003; 284(1): R1 - R12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Duchene, J. P. Schanstra, C. Pecher, A. Pizard, C. Susini, J.-P. Esteve, J.-L. Bascands, and J.-P. Girolami A Novel Protein-Protein Interaction between a G Protein-coupled Receptor and the Phosphatase SHP-2 Is Involved in Bradykinin-induced Inhibition of Cell Proliferation J. Biol. Chem., October 18, 2002; 277(43): 40375 - 40383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. T. Massa, S. L. Ropka, S. Saha, K. L. Fecenko, and K. L. Beuler Critical Role for Protein Tyrosine Phosphatase SHP-1 in Controlling Infection of Central Nervous System Glia and Demyelination by Theiler's Murine Encephalomyelitis Virus J. Virol., July 17, 2002; 76(16): 8335 - 8346. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
