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Department of Molecular Medicine (P.T.-E., A.F.-M., G.N.), Karolinska Institutet, Karolinska Hospital, 171 76 Stockholm, Sweden, Department of Woman and Child Health (A.S.-E., L.S.), Karolinska Institutet, Division for Reproductive Endocrinology, Karolinska Hospital, 171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: Petra Tollet-Egnell, Department of Molecular Medicine, Karolinska Institutet, CMM L8:01, Karolinska Hospital, 171 76 Stockholm, Sweden. E-mail: petra.tollet.egnell{at}molmed.ki.se
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH activation of the JAK-STAT pathway is short-lived in nature; e.g. in cell lines transfected with the GHR, gel retardation analysis (EMSA) indicates that phosphorylated STAT5 is transiently activated during approximately 30 min in cells stimulated by GH (19). Interestingly, the timespan during which STATs are activated can be prolonged by inhibition of protein synthesis (19), indicating that proteins with the capacity to "shut off" GHR signal transduction might exist. This concept has recently been substantiated by the discovery of the SOCS (suppressor of cytokine signaling) proteins (20, 21, 22, 23, 24, 25, 26).
At least eight members of the SOCS family exist (SOCS-1 to SOCS-7 and CIS, where CIS stands for cytokine-inducible SH2-containing protein) (27), and they appear to form part of a negative feedback loop that regulates cytokine signal transduction (28, 29). Cytokine-induced expression of the SOCS genes has been reported to occur with different kinetics, and once produced, the various members of the SOCS family appear to inhibit signaling in different ways. Structurally, the SOCS proteins contain a common central SH2 domain and a novel C-terminal motif called the SOCS box. Although the function of the SOCS box remains unknown, a model has been proposed in which the SOCS box acts as a kinase inhibitor and the SH2 domain dictates with which member of the JAK family of protein tyrosine kinases each protein interacts. However, the different SOCS family members do not display a uniform mechanism of action. SOCS-1, which is induced in response to a range of cytokines, was recently shown to arrest cytokine signaling by binding to and inhibiting the intrinsic activity of four different JAK kinases (26, 30). In contrast, CIS does not interact with JAK (25). It has instead been suggested that CIS acts by competing with signaling molecules such as the STATs for binding to specific phosphorylated tyrosine residues within the Epo and GM-CSF-ß receptors (20, 31).
Variable levels of SOCS expression may be the keys to understanding the molecular mechanisms behind differential cellular responsiveness to cytokines. Several physiological situations exist where the sensitivity to GH is altered, including aging, severe GH deficiency and Cushing’s syndrome (1, 32, 33, 34), and it is therefore relevant to study SOCS expression in relation to GH actions. SOCS-1, SOCS-2, SOCS-3 and CIS messenger RNA (mRNA) were recently reported to be induced by GH in 3T3-F442A fibroblasts (35). Furthermore, SOCS-1 and SOCS-3 were shown to inhibit GH stimulated transcription of the GH-responsive serine protease inhibitor 2.1 gene promoter. In the present study, we have investigated the effects of aging and hypophysectomy on SOCS expression in different rat tissues. The effects of GH on SOCS-2, SOCS-3, and CIS mRNA levels were determined in hypophysectomized or intact rats. GH regulation of hepatic SOCS expression was further characterized in primary cultures of rat hepatocytes. Finally, the effect of glucocorticoids on hepatic SOCS expression was investigated in vivo and in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Collagenase (type IV) and insulin were obtained from Sigma Chemical Co. (St Louis, MO). Proteinase K was purchased from
Merck & Co., Darmstadt, Germany), Biomatrix-EHS, RNase-A
and RNase-T1 from Boehringer Ingelheim Bioproducts Partnership (Heidelberg, Germany), and glass-fiber filters
(Whatman GF/C) from Whatman Ltd. (Madison,
Kent, UK). Reagents for in vitro transcription were obtained
from Promega Corp. Biotech (Madison, WI).
Cell cultures
Rat hepatocytes were isolated and cultured on matrigel
(Biomatrix-EHS) in Williams E medium supplemented with insulin (1
µg/ml), Na2SeO3 (0.1 µM),
vitamin C (30 mM), penicillin (50 U/ml), streptomycin (50
µg/ml), essentially as described previously (36). Hormonal treatments
were started at approximately 40 h of culture age. At harvesting
of cells, the medium was aspirated from the plates, cells were washed
in PBS and scraped off with a rubber spatula in 1 x SET (1%
(wt/vol) SDS, 10 mM EDTA, and 20 mM Tris-HCl,
pH 7.5).
Analysis of mRNA expression
Total nucleic acids (TNA) were isolated by homogenization of
tissue specimens, using a polytrone PT-2000 (Kinematica AG, Littau,
Switzerland). Digestion of samples with proteinase K and
subsequent extraction with chloroform and phenol have been described
previously (37). When TNA samples were prepared from cultured
hepatocytes, pooled cells from four to five culture dishes were lysed
in 1 x SET and further processed as described above. mRNA levels
corresponding to SOCS-2, SOCS-3 and CIS expression were measured in TNA
samples, using a solution hybridization/RNase protection assay.
Transcript-specific [35S]UTP-labeled cRNA probes were
transcribed in vitro from the respective complementary DNA
(cDNA) vector construct, according to the method of Melton et
al. (38). The probes that were used to detect these transcripts
were isolated from rat genomic DNA, using PCR technique with the
following oligonucleotide primers, designed to amplify specific regions
within the SOCS genes.
SOCS-2 Sense primer: 5' GAG CTC AGT CAA ACA GGA TGG TAC T 3'
Antisense primer: 5' AGA ATC CAA TCT GAA TTT CCC ATC T 3'
PCR product length: 201 bases
SOCS-3 Sense primer: 5' GAG TAC CCC CAA GAG AGC TTA CTA C 3'
Antisense Primer: 5' CTC CTT AAA GTG GAG CAT CAT ACT G 3'
PCR product length: 209 bases
CIS Sense Primer: 5' ATC TTG TCC TTT GCT GGC TGT 3'
Antisense Primer: 5' CCC GAA GGT AGG AGA ACG TCT 3'
PCR product length: 215 bases
The obtained PCR products were blunt-cloned into the BamHI site of Bluescript SK (Stratagene, La Jolla, CA), followed by sequencing of both strands by the method of dideoxy chain termination. The sequences are available from the authors on request. mRNA levels corresponding to glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (39), insulin-like growth factor (IGF)-I (40), GHR (41), and CYP2C12 (42) expression were measured using specific [35S]UTP-labeled cRNA probes, as described previously. Quantitations of specific mRNA species were achieved by comparison with a standard curve obtained from hybridizations to known amounts of in vitro synthesized mRNA. The concentration of nucleic acids in TNA samples was measured spectrometrically. Samples were analyzed in triplicate and the results are expressed as fg specific mRNA per µg TNA. To permit accurate comparison of specific mRNA levels between different tissues or treatments, the samples of interest were always analyzed in the same assay at the same time. The data presented were statistically analyzed, using Students t test or Kruskal-Wallis test with significance evaluated using Dunn’s method. Obtained P values are presented in the figure legends.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). A clear differential expression
of SOCS-2, SOCS-3, and CIS mRNAs was noted in different tissues. SOCS-2
expression was high in liver and heart, whereas SOCS-3 mRNA was found
at high levels in lung and spleen. CIS expression was high in kidney,
skeletal muscle, and adipose tissue but was also found in relatively
high levels in other tissues. Liver expressed high levels of SOCS-2
compared with muscle, whereas CIS and SOCS-3 expression was in the same
range in liver and muscle. Due to the relative ease of SOCS detection
in liver, muscle, and fat, these tissues were selected for further
studies. It is well known that GH regulates various important metabolic
functions in liver, muscle, and adipose tissue (2, 3, 4). These metabolic
functions have altered GH sensitivity in hypophysectomized (Hx) or aged
animals (32, 33, 43).
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). In contrast, SOCS-3 mRNA levels
were not altered in liver and muscle, whereas adipose tissue from old
rats displayed a 5-fold higher level of SOCS-3 mRNA compared with young
rats. Aging reduced the expression of CIS mRNA in all tissues
investigated. Thus, both increased and decreased SOCS mRNA levels were
observed.
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, hypophysectomy of male rats caused a dramatic reduction in hepatic
SOCS-2 and CIS expression, whereas no effect was seen on SOCS-3 mRNA
levels. A similar type of pituitary dependence of SOCS-2 and CIS was
observed in muscle and adipose tissue. No major sex-differences in SOCS
expression were observed in these tissues (Fig. 3). Moreover, as
demonstrated in Fig. 4, hypophysectomy of
female rats led to the same reduction of SOCS-2 expression as observed
in male rats.
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, GH treatment restored hepatic SOCS-2 mRNA expression, albeit
not entirely to the levels detected in intact females. However, when
two injections of hGH per day were given in addition to infusion of the
hormone, a complete restoration of SOCS-2 expression was observed
(intact female: 794.1 ± 85.0 fg mRNA/µg TNA, Hx female treated
with two injections of hGH per day in addition to infusion of the
hormone: 798.9 ± 121.0 fg mRNA/µg TNA). IGF-I mRNA levels were
analyzed in the same samples, and a similar type of GH dependence was
observed for the two different gene products (Fig. 4). The infusion of
GH restored animal weight to 86% of intact rats (data not shown),
further indicating that the hormonal treatment had been sufficient.
These results indicate that GH is an important regulator of SOCS-2
expression in rat liver.
Glucocorticoids do not affect hepatic SOCS-2 expression in Hx
rats
Supraphysiological levels of glucocorticoids, whether endogenous
(Cushing’s syndrome) or exogenous (glucocorticoid therapy), inhibit
growth in children and immature animals (34, 44). The mechanisms of
interaction between glucocorticoids and somatotropic hormones on the
cellular and molecular level are poorly understood. However, the
synthetic glucocorticoid dexamethasone (dex) has been shown to inhibit
the ability of GH to elicit several early events in GH signaling and
gene regulation in cultured cells (36, 45, 46), including inhibition of
GHR expression, reduced GH binding to GHR, decreased GH-induced tyrosyl
phosphorylation of GHR and JAK2, and reduced expression of the IGF-I
and CYP2C12 genes. To investigate whether this negative effect of
glucocorticoids might be mediated by increased cellular levels of SOCS
proteins, Hx rats were treated by infusion from osmotic minipumps for 1
week with dex, in the absence or presence of GH. As expected, both
IGF-I mRNA levels (Fig. 4) and animal weight (data not shown) were
reduced in animals treated with GH in combination with dex, compared
with rats treated with GH alone. The same inhibitory effect by dex was
observed on GH-induced SOCS-2 expression. However, dex treatment did
not affect the basal expression of SOCS-2 mRNA, suggesting that other
cellular changes, e.g. a disturbance at the level of the
receptor, are more likely to explain the steroid-induced GH resistance.
Indeed, GHR mRNA levels were reduced in animals treated with GH in
combination with dex (0.50 ± 0.03 pg mRNA/µg TNA), compared
with rats treated with GH alone (2.32 ± 0.14 pg mRNA/µg TNA).
These results do not exclude that dex could have a more rapid effect on
SOCS expression, which might affect GH sensitivity.
GH induces SOCS expression in primary cultures of rat
hepatocytes
To determine whether the GH-induced SOCS expression in Hx rats was
mediated through a direct action on the cell, primary cultures of rat
hepatocytes were used to study hepatic SOCS expression. Dose-response
experiments were carried out at different time-points after GH addition
to cultured cells. As demonstrated in Fig. 5, SOCS-2 (Fig. 5A), CIS (Fig. 5 A and B)
and SOCS-3 (Fig. 5B) mRNA levels were increased by GH treatment in a
dose-dependent manner. The dose-response curves for SOCS mRNA
expression were shown to be very similar to previously reported
dose-response curves for other GH regulated genes in this cell-system
(36). SOCS-2 expression was studied after 24 h of GH treatment,
and a maximum effect of GH was reached between 0.5 and 5.0 µg/ml
(Fig. 5A). The SOCS-3 dose-response experiment was carried out at an
earlier time-point because the expression of SOCS-3 has been reported
to be more rapid (35). As demonstrated in Fig. 5B, 1 h of GH
treatment led to a bell-shaped dose-response curve with its maximum
between 0.1 and 1.0 µg/ml. The bell-shaped curve might be consistent
with a dimerization model for the GH receptor (7, 8). CIS expression
was highly induced by GH at both time points. As shown in Fig. 5, A and
B, the two CIS dose-response curves had the same shape at the different
time points but differed from the curves obtained for SOCS-2 and
SOCS-3. The maximum effect on CIS expression was reached between 0.5
and 5.0 µg/ml at both time points. CIS was more sensitive to lower
concentrations of GH, compared with SOCS-2. However, SOCS-3 seemed to
be the most sensitive gene, with a 3-fold induced expression in cells
treated with 1 ng hGH/ml. These results led us to perform the
subsequent studies on GH effects on SOCS expression using a dose of 0.5
µg/ml.
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. Significantly induced levels of SOCS-2
mRNA started to appear after 30 min of GH treatment and increased with
time throughout the experiment. SOCS-2 mRNA levels were increased about
20-fold at 24 h after hGH addition. The GH-induced expression of
SOCS-3 was rapid and transient, with mRNA levels peaking at 30 min of
treatment. The time-course of CIS mRNA expression showed both an early
peak at 60 min, as well as a steady increase of mRNA between 4 and
24 h after hGH addition. The maximum effect induced by GH on these
two genes was six- and 10-fold, respectively. The pattern of SOCS
expression in this study, using primary cultures of rat hepatocytes, is
similar to previously demonstrated GH-induced SOCS expression in
3T3-F442A fibroblasts (35). Taken together with the data presented in
Fig. 5, A and B, these results indicate that the molecular mechanism
behind GH-regulation of SOCS expression might be different for the
different SOCS genes.
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and 6). The GH-induced levels of SOCS-2 and CIS were comparable to
levels observed in intact animals, suggesting that GH is an important
regulator of these genes in rat liver. SOCS-3 mRNA levels were not
different between untreated cultured hepatocytes and intact livers,
which is in line with the finding that hepatic SOCS-3 expression was
unaltered in the Hx animals (Fig. 3).
Glucocorticoids potentiate GH induced SOCS-2 and CIS mRNA
expression
As described above, GH-induced hepatic expression of SOCS-2 was
blocked by dex treatment in Hx rats (Fig. 4). To determine whether a
similar inhibitory effect on SOCS expression was apparent in
vitro, cultured hepatocytes were treated with dex in the absence
or presence of GH. As indicated in Fig. 7, dex (100 nM) for 1 or
24 h did not affect the basal levels of any SOCS mRNA analyzed.
However, the GH-induced expression of SOCS-2 and CIS was significantly
higher at both time-points. In line with a previous study (36), the
GH-induced expression of IGF-I and CYP2C12 was reduced in cells treated
with GH in combination with dex, compared with cells treated with GH
alone (Fig. 7). These results indicate a possible role for SOCS in
mediating the negative action of glucocorticoids on different actions
of GH. Although dex did not increase hepatic SOCS-2 mRNA levels in
GH-treated Hx rats, due to reduced levels of GHR, it is evident that
dex could have a more rapid effect on SOCS-2 and CIS expression in the
isolated hepatocyte. The discrepancy between in vivo and
in vitro results could be explained by differences in dose
or duration of the hormonal treatments.
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, both the basal and the GH-induced
expression of SOCS-3 were inhibited in the presence of dex. Thus, both
positive and negative effects were observed, depending on the SOCS-gene
analyzed.
GH down-regulates hepatic SOCS-2 expression in old rats
If SOCS proteins were involved in the desensitization of
peripheral tissues toward GH, the expression of GH-regulated genes
would be more sensitive toward GH in tissues with decreased SOCS
expression. To test this hypothesis, both old (at 2 years of age) and
younger (at 8 weeks of age) rats were infused with GH for 3 weeks,
tissues removed and analyzed for SOCS expression and GH sensitivity. As
expected, the SOCS-2 mRNA expression was reduced in livers from old
rats. No GH-dependent changes in either SOCS-2 or IGF-I mRNA levels
could be detected in younger rats (Fig. 8). The inability of young rats to
respond to GH in terms of IGF-I mRNA was expected in light of the
presence of high levels of endogenous GH in intact young males. Another
GH responsive gene, CYP2C12 (47), was induced 16-fold in the same
samples, indicating that the GH treatment of these rats had been
sufficient. Unlike the IGF-I gene, expression of the CYP2C12 gene is
female-specific in rat liver (48). CYP2C12 is induced by the
female-specific pattern of GH secretion, which is mimicked by
continuous infusion of GH. CYP2C12 expression is therefore a sensitive
marker of GH responsiveness in male rat liver. Surprisingly, when liver
samples from the old rats were analyzed, a significant GH-dependent
reduction of SOCS-2 mRNA was observed (Fig. 8). As in the young rats,
IGF-I mRNA expression was not altered by GH treatment, whereas CYP2C12
was highly induced. The GH responsiveness of this gene was about 4
times higher (60-fold) as compared with younger rats. The repressing
effect of GH on SOCS-2 mRNA levels was also observed in muscle and
adipose tissue of old animals and appeared to be specific to SOCS-2
(data not shown). In summary, these results indicate that SOCS mRNA
levels may correlate to GH sensitivity of specific GH-regulated
cellular functions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because SOCS-3 has been shown to block transcriptional actions of GH (35), it might be anticipated to be expressed at low levels in classical GH target tissues such as liver, skeletal muscle, and adipose tissue. Interestingly, SOCS-3 mRNA levels were demonstrated to be increased 5-fold in adipose tissue of old rats. It is tempting to speculate that increased constitutive levels of SOCS-3 protein in fat might explain the observed age-dependent decrease in GH sensitivity and subsequent changes in body composition, characteristic of aged animals, and man. The nature of the factor(s) regulating the observed increase in SOCS-3 mRNA in adipose tissue is not known. Leptin is one out of several cytokines that have been shown to increase SOCS-3 expression (50). Leptin is secreted by adipose tissue, and the plasma levels are highly correlated with adipose tissue mass (51). Because old animals are known to have a greater fat mass compared with younger (52), it could be assumed that the aged rats in our study had higher plasma levels of leptin compared with the younger rats. Under the assumption and that the aged rats had higher plasma levels of leptin, and that leptin receptors are expressed in adipocytes, leptin would be a possible candidate as to the identity of the SOCS-3 inducing agent in fat from old rats. Further studies are however needed to test this hypothesis.
The secretion of GH from the pituitary is reduced during aging. The reduced expression of SOCS-2 and CIS mRNA observed in old rats could be due to reduced plasma GH levels because SOCS-2 and CIS expression was shown to be dependent on the pituitary and to be induced by GH in primary cultures of rat hepatocytes. GH appeared to be an important positive regulator of hepatic SOCS-2 and CIS expression in younger animals. However, when old rats were treated with GH for 3 weeks, SOCS-2 expression was reduced. Neither CIS nor SOCS-3 expression was affected by the hormonal treatment (data not shown). It thus appears as if other factors interact with GH to determine whether a positive (as in young rats) or a negative (as in old rats) effect on SOCS-2 expression is obtained.
If SOCS-2 were involved in the desensitization of tissues toward GH, then the hepatic expression of GH-regulated genes would be increased in old (compared with young) rats treated with GH. Indeed, as demonstrated here, the hepatic expression of CYP2C12 was superinduced in old rats after treatment with GH for 3 weeks. GH did not induce another GH-regulated gene, IGF-I, in these experiments, indicating that distinct effects of GH are differently modulated during aging. This could be explained by the assumption that different signaling molecules mediate distinct cellular effects of GH, and that the SOCS proteins might affect these factors in a dissimilar way.
It is well known that severe GH deficiency is coupled to a state of GH hypersensitivity, and that this hypersensitivity is reduced by GH treatment (1, 32). Thus, the reduced expression of SOCS-2 and CIS in hypophysectomized rats is another indication of SOCS proteins acting as desensitizers of GH action. The GH-dependent decrease in GH sensitivity in these animals could be explained by the fact that GH treatment will lead to induced levels of SOCS proteins. In this regard, it is relevant to point out that SOCS-2 and CIS were both induced in isolated hepatocytes by GH in a relatively slow and continuous fashion, which is compatible with SOCS-2 and CIS being markers of reduced GH sensitivity. It was, however, recently reported that SOCS-2 and CIS (in contrast to SOCS-1 and SOCS-3) are unable to inhibit GH-stimulated transcription of the GH responsive serine protease inhibitor 2.1 gene promoter. This does not exclude a role for SOCS-2 and CIS as desensitizers of other cellular effects of GH. Further studies are required to elucidate the exact significance of reduced levels of SOCS-2 and CIS in GH deficient animals.
SOCS-3 mRNA levels were not reduced upon removal of the pituitary and showed completely different kinetics when induced by GH in cultured hepatocytes. The rapid and short-lived increase in SOCS-3 mRNA levels is more compatible with a transient insensitivity toward GH. In this respect, it is relevant to point out that differential GH sensitivity can be regulated on an hourly basis. The insulin-like acute effects of GH observed in adipose tissue (53) are dependent on the absence of GH for 2–3 h. It is tempting to speculate that GH-induced SOCS-3 protein is causing this lag-phase of GH response.
Several different hepatic functions are regulated by the secretory pattern of GH in rodents (4), where male rats secrete GH in a pulsatile fashion, with peaking levels of plasma GH every third hour, compared with females with a more continuous GH secretion (54). Furthermore, it has been reported by Waxman and colleagues (55), that liver STAT5 is uniquely responsive to the temporal pattern of GH secretion: intermittent plasma GH pulses trigger rapid and repeated tyrosine phosphorylation and nuclear translocation of liver STAT5, while continuous plasma GH exposure leads to desensitization of this pathway. To determine such a type of differential GH sensitivity of signaling molecules and subsequent activation of target genes, a rapid regulation of e.g. SOCS-3 seems relevant. If this hypothesis is correct, the endogenous expression of SOCS-3 should be pulsatile in male rats and more continuous in female rats. The results presented here showed no sex-difference in SOCS-3 mRNA levels, speaking against such a hypothesis. It is, however, possible that a more careful study, with a larger group of animals, would have given results showing a more sexually differentiated expression of SOCS-3 mRNA. It should also be possible to determine whether a pulsatile administration of GH to GH-deficient animals or isolated cells will induce a pulsatile expression of SOCS-3.
Interestingly, GH has also been shown to activate STAT1 and STAT3 in liver tissue, but with a dependence on the temporal plasma profile that is distinct from STAT5, and with a striking desensitization following a single hormone pulse that is not observed with liver STAT5 (56). STAT1 and STAT3, but not liver STAT5, was reported to become desensitized with respect to tyrosine phosphorylation and DNA binding in response to repeated cycles of GH pulsation. It is tempting to speculate that GH-induced levels of SOCS proteins, like SOCS-2 or CIS, might play a role in mediating this desensitization. Further studies are required to test this hypothesis.
The synthetic glucocorticoid dexamethasone (dex) has been shown to
antagonize the growth-promoting action of GH in peripheral tissues. In
the present study, we found no evidence for a direct steroid regulation
of the hepatic SOCS expression. However, dex treatment potentiated the
GH-induced expression of SOCS-2 and CIS in primary cultures of rat
hepatocytes, indicating a potential role for SOCS proteins in
dex-mediated GH insensitivity. King et al. (45) have
reported that dex down-regulates GH binding to GHR, and it could be
speculated that this inhibitory effect is mediated through dex-induced
levels of SOCS protein. It is not yet known how GH activates
transcription of the different SOCS genes, although it has been
suggested that STAT3 mediates transcription of the SOCS-1 gene (22),
and that STAT5 regulates transcription of the CIS gene (31). However,
dex-activated glucocorticoid receptors (GR) might interact with
GH-activated transcription factors, such as STATs, leading to a
potentiation of the GH response. Glucocorticoids have recently been
shown to affect milk protein gene expression via association of the GR
with STAT5 (57). Similarly, a synergistic activation by GR and STAT3,
of the IL-6 response element on the rat 
2-macroglobulin promoter,
was suggested by Takeda et al. (58). Thus, there is a
functional coupling between STAT-dependent and GR-dependent gene
transcription, although the exact molecular mechanism of the cross-talk
has not yet been elucidated (59). A dex-dependent increase in SOCS
protein would then lead to reduced GH binding and/or signaling from
GHR, decreased levels of GH-regulated gene products, and to impaired
animal growth. GH-mediated cellular effects would also be reduced due
to glucocorticoid-mediated down-regulation of GHR mRNA and protein.
Taken together, the results presented here show that the hormonal status of an animal could affect the expression of SOCS proteins and support a concept that SOCS expression could influence hormonal responsiveness of the animal. We found that SOCS mRNA levels were dependent on the pituitary as well as influenced by animal age, GH, and glucocorticoid treatments. Both the basal levels of SOCS expression and the effects of GH and aging were shown to be different in different tissues. Furthermore, some of the effects were specific to the different SOCS genes. Variable levels of specific SOCS expression may therefore play a role in the molecular mechanism behind differential cellular responsiveness to cytokines. This study was limited to the investigation of SOCS-2, SOCS-3, and CIS, and the pattern of cellular SOCS expression is therefore likely to be even more complex. The SOCS proteins do not seem to use a uniform mechanism of action, and among several questions to be answered is the nature of molecular interactions that SOCS-2, SOCS-3, and CIS are triggering in the cell. Overall, we believe that a closer study of the exact interaction of different SOCS proteins with the GH receptor signaling machinery could give valuable clues to the regulation of GH receptor function and GH sensitivity.
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Acknowledgments |
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Footnotes |
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Received November 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S Eleswarapu and H Jiang Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver J. Endocrinol., January 1, 2005; 184(1): 95 - 105. [Abstract] [Full Text] [PDF]
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P. Kotokorpi, C. Gardmo, C. S. Nystrom, and A. Mode Activation of the Glucocorticoid Receptor or Liver X Receptors Interferes with Growth Hormone-Induced akr1b7 Gene Expression in Rat Hepatocytes Endocrinology, December 1, 2004; 145(12): 5704 - 5713. [Abstract] [Full Text] [PDF]
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J. Rieusset, J. Seydoux, S. I. Anghel, P. Escher, L. Michalik, N. Soon Tan, D. Metzger, P. Chambon, W. Wahli, and B. Desvergne Altered Growth in Male Peroxisome Proliferator-Activated Receptor {gamma} (PPAR{gamma}) Heterozygous Mice: Involvement of PPAR{gamma} in a Negative Feedback Regulation of Growth Hormone Action Mol. Endocrinol., October 1, 2004; 18(10): 2363 - 2377. [Abstract] [Full Text] [PDF]
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J. G. Miquet, A. I. Sotelo, A. Bartke, and D. Turyn Suppression of Growth Hormone (GH) Janus Tyrosine Kinase 2/Signal Transducer and Activator of Transcription 5 Signaling Pathway in Transgenic Mice Overexpressing Bovine GH Endocrinology, June 1, 2004; 145(6): 2824 - 2832. [Abstract] [Full Text] [PDF]
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 J. Woelfle and P. Rotwein In vivo regulation of growth hormone-stimulated gene transcription by STAT5b Am J Physiol Endocrinol Metab, March 1, 2004; 286(3): E393 - E401. [Abstract] [Full Text] [PDF]
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 P. Tollet-Egnell, P. Parini, N. Stahlberg, I. Lonnstedt, N. H. Lee, M. Rudling, A. Flores-Morales, and G. Norstedt Growth hormone-mediated alteration of fuel metabolism in the aged rat as determined from transcript profiles Physiol Genomics, January 15, 2004; 16(2): 261 - 267. [Abstract] [Full Text] [PDF]
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V. C. Calegari, R. M. N. Bezerra, M. A. Torsoni, A. S. Torsoni, K. G. Franchini, M. J. A. Saad, and L. A. Velloso Suppressor of Cytokine Signaling 3 Is Induced by Angiotensin II in Heart and Isolated Cardiomyocytes, and Participates in Desensitization Endocrinology, October 1, 2003; 144(10): 4586 - 4596. [Abstract] [Full Text] [PDF]
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S. Faderl, D. Harris, Q. Van, H. M. Kantarjian, M. Talpaz, and Z. Estrov Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia Blood, July 15, 2003; 102(2): 630 - 637. [Abstract] [Full Text] [PDF]
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S. Welle, A. I. Brooks, J. M. Delehanty, N. Needler, and C. A. Thornton Gene expression profile of aging in human muscle Physiol Genomics, July 7, 2003; 14(2): 149 - 159. [Abstract] [Full Text] [PDF]
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L. Q. Hong-Brown, C. R. Brown, R. N. Cooney, R. A. Frost, and C. H. Lang Sepsis-induced muscle growth hormone resistance occurs independently of STAT5 phosphorylation Am J Physiol Endocrinol Metab, July 1, 2003; 285(1): E63 - E72. [Abstract] [Full Text] [PDF]
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L. Du, G. P. Frick, L.-R. Tai, A. Yoshimura, and H. M. Goodman Interaction of the Growth Hormone Receptor with Cytokine-Induced Src Homology Domain 2 Protein in Rat Adipocytes Endocrinology, March 1, 2003; 144(3): 868 - 876. [Abstract] [Full Text] [PDF]
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 K. C. Leung, N. Doyle, M. Ballesteros, K. Sjogren, C. K. W. Watts, T. H. Low, G. M. Leong, R. J. M. Ross, and K. K. Y. Ho Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2 PNAS, February 4, 2003; 100(3): 1016 - 1021. [Abstract] [Full Text] [PDF]
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C. J. Greenhalgh, D. Metcalf, A. L. Thaus, J. E. Corbin, R. Uren, P. O. Morgan, L. J. Fabri, J.-G. Zhang, H. M. Martin, T. A. Willson, et al. Biological Evidence That SOCS-2 Can Act Either as an Enhancer or Suppressor of Growth Hormone Signaling J. Biol. Chem., October 18, 2002; 277(43): 40181 - 40184. [Abstract] [Full Text] [PDF]
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P. Ribaux, A. Gjinovci, H. B. Sadowski, and P. B. Iynedjian Discrimination between Signaling Pathways in Regulation of Specific Gene Expression by Insulin and Growth Hormone in Hepatocytes Endocrinology, October 1, 2002; 143(10): 3766 - 3772. [Abstract] [Full Text] [PDF]
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O. Morales, M. H. Faulds, U. J. Lindgren, and L.-A. Haldosen 1alpha ,25-Dihydroxyvitamin D3 Inhibits GH-induced Expression of SOCS-3 and CIS and Prolongs Growth Hormone Signaling via the Janus Kinase (JAK2)/Signal Transducers and Activators of Transcription (STAT5) System in Osteoblast-like Cells J. Biol. Chem., September 13, 2002; 277(38): 34879 - 34884. [Abstract] [Full Text] [PDF]
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S. G. Ronn, J. A. Hansen, K. Lindberg, A. E. Karlsen, and N. Billestrup The Effect of Suppressor of Cytokine Signaling 3 on GH Signaling in {beta}-Cells Mol. Endocrinol., September 1, 2002; 16(9): 2124 - 2134. [Abstract] [Full Text] [PDF]
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 S. F. Soriano, P. Hernanz-Falcon, J. M. Rodriguez-Frade, A. M. de Ana, R. Garzon, C. Carvalho-Pinto, A. J. Vila-Coro, A. Zaballos, D. Balomenos, C. Martinez-A., et al. Functional Inactivation of CXC Chemokine Receptor 4-mediated Responses through SOCS3 Up-regulation J. Exp. Med., August 5, 2002; 196(3): 311 - 321. [Abstract] [Full Text] [PDF]
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C. J. Greenhalgh, P. Bertolino, S. L. Asa, D. Metcalf, J. E. Corbin, T. E. Adams, H. W. Davey, N. A. Nicola, D. J. Hilton, and W. S. Alexander Growth Enhancement in Suppressor of Cytokine Signaling 2 (SOCS-2)-Deficient Mice Is Dependent on Signal Transducer and Activator of Transcription 5b (STAT5b) Mol. Endocrinol., June 1, 2002; 16(6): 1394 - 1406. [Abstract] [Full Text] [PDF]
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V. Beauloye, B. Willems, V. de Coninck, S. J. Frank, M. Edery, and J.-P. Thissen Impairment of Liver GH Receptor Signaling by Fasting Endocrinology, March 1, 2002; 143(3): 792 - 800. [Abstract] [Full Text] [PDF]
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L. Gonzalez, J. G. Miquet, A. I. Sotelo, A. Bartke, and D. Turyn Cytokine-Inducible SH2 Protein Up-Regulation Is Associated with Desensitization of GH Signaling in GHRH-Transgenic Mice Endocrinology, February 1, 2002; 143(2): 386 - 394. [Abstract] [Full Text] [PDF]
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C. Ling and H. Billig PRL Receptor-Mediated Effects in Female Mouse Adipocytes: PRL Induces Suppressors of Cytokine Signaling Expression and Suppresses Insulin-Induced Leptin Production in Adipocytes in Vitro Endocrinology, November 1, 2001; 142(11): 4880 - 4890. [Abstract] [Full Text] [PDF]
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 C. J. Greenhalgh and D. J. Hilton Negative regulation of cytokine signaling J. Leukoc. Biol., September 1, 2001; 70(3): 348 - 356. [Abstract] [Full Text] [PDF]
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A. Flores-Morales, L. Fernandez, E. Rico-Bautista, A. Umana, C. Negrin, J.-G. Zhang, and G. Norstedt Endoplasmic Reticulum Stress Prolongs GH-Induced Janus Kinase (JAK2)/Signal Transducer and Activator of Transcription (STAT5) Signaling Pathway Mol. Endocrinol., September 1, 2001; 15(9): 1471 - 1483. [Abstract] [Full Text] [PDF]
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C. L. Sadowski, T. T. Wheeler, L.-H. Wang, and H. B. Sadowski GH Regulation of IGF-I and Suppressor of Cytokine Signaling Gene Expression in C2C12 Skeletal Muscle Cells Endocrinology, September 1, 2001; 142(9): 3890 - 3900. [Abstract] [Full Text] [PDF]
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 P. L. Bergad, S. J. Schwarzenberg, J. T. Humbert, M. Morrison, S. Amarasinghe, H. C. Towle, and S. A. Berry Inhibition of growth hormone action in models of inflammation Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1906 - C1917. [Abstract] [Full Text] [PDF]
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A. Colson, A. Le Cam, D. Maiter, M. Edery, and J.-P. Thissen Potentiation of Growth Hormone-Induced Liver Suppressors of Cytokine Signaling Messenger Ribonucleic Acid by Cytokines Endocrinology, October 1, 2000; 141(10): 3687 - 3695. [Abstract] [Full Text] [PDF]
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M. R. Stofega, J. Herrington, N. Billestrup, and C. Carter-Su Mutation of the SHP-2 Binding Site in Growth Hormone (GH) Receptor Prolongs GH-Promoted Tyrosyl Phosphorylation of GH Receptor, JAK2, and STAT5B Mol. Endocrinol., September 1, 2000; 14(9): 1338 - 1350. [Abstract] [Full Text]
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P. A. Ram and D. J. Waxman SOCS/CIS Protein Inhibition of Growth Hormone-stimulated STAT5 Signaling by Multiple Mechanisms J. Biol. Chem., December 10, 1999; 274(50): 35553 - 35561. [Abstract] [Full Text] [PDF]
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P. A. Ram and D. J. Waxman Role of the Cytokine-inducible SH2 Protein CIS in Desensitization of STAT5b Signaling by Continuous Growth Hormone J. Biol. Chem., December 8, 2000; 275(50): 39487 - 39496. [Abstract] [Full Text] [PDF]
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