Defective Retinoic Acid Regulation of the Pit-1 Gene Enhancer: A Novel Mechanism of Combined Pituitary Hormone Deficiency

Laurie E. Cohen, Kerstin Zanger, Thierry Brue, Fredric E. Wondisford and Sally Radovick

Divisions of Endocrinology Departments of Medicine Children’s Hospital (L.E.C., K.Z., T.B., S.R.) and Beth Israel Deaconess Medical Center (F.E.W.), Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-ß subunit (TSH-ß), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit-1 (whose official nomenclature is now POU1F1) is a member of a family (POU) of transcription factors regulating mammalian development. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for high-affinity DNA binding on the GH and PRL genes (1). When bound to DNA, Pit-1 activates GH and PRL gene expression, in part, through an N-terminal transactivation domain rich in hydroxylated amino acid residues (2, 3). During development, Pit-1 gene expression precedes GH and PRL gene expression in the somatotroph and lactotroph, respectively, and is thought to be the major cell-specific activator of hormone expression from these cell types (4, 5, 6, 7, 8, 9). Additional nuclear factors, however, appear to be necessary for expression of the GH and PRL genes. Lipkin et al. (10) cloned a zinc finger transcription factor, termed Zn-15, which is responsible with Pit-1 for synergistic activation of the GH gene. In addition, some investigators suggest that Pit-1 synergistically activates the GH gene in the presence of thyroid hormone receptors (11). Other investigators, however, have not confirmed this finding (12). Likewise, the estrogen receptor is required, along with Pit-1, for PRL gene distal enhancer activation by estradiol (13, 14, 15).

Rhodes et al. (16) explored the molecular mechanism responsible for activation of the Pit-1 gene in vivo. They demonstrated that an enhancer element, located more than 10 kb upstream of the transcriptional start site, was essential for pituitary-specific expression of the Pit-1 gene in transgenic mice. Like the GH and PRL genes, Pit-1 alone was not sufficient to direct pituitary-specific expression of its own gene via this distal enhancer element. In addition to several Pit-1 DNA-binding sites, this enhancer contains at least two retinoic acid response elements (RAREs). Interestingly, the retinoic acid receptor (RAR) functionally interacts with Pit-1 on one of these latter elements to mediate a synergistic activation of the Pit-1 gene in response to retinoic acid (RA). It is intriguing that a developmental factor (Pit-1) and a morphogen (RA) would interact at this element and suggests that RA may play a critical role in pituitary development.

Point mutations in the Pit-1 gene have been found in association with combined pituitary hormone deficiency (CPHD) of GH, PRL, and TSH (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), some on only one allele (19, 21, 22). Unlike the autosomal recessive mutations in which loss of function from one allele can not cause CPHD, the molecular mechanism of the sporadic mutations is unclear. One mutation (R271W) dominantly inhibits activation of the GH and PRL genes by wild-type (wt) Pit-1, and its properties have been described elsewhere (19, 22).

In the present report, we have explored the mechanism of CPHD in a patient with a novel Pit-1 gene mutation present on only one allele. This mutation (K216E) does not inhibit activation of the GH and PRL target genes. It, however, blocks RA induction via the Pit-1 gene distal enhancer. Our results indicate that Pit-1 gene mutations can cause CPHD by selectively inhibiting gene activation by the superfamily of nuclear hormone receptors.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Novel Pit-1 Gene Mutation in a Patient with CPHD
A patient was evaluated at Children’s Hospital for short stature. The patient had measurable GH levels, but insulin-induced hypoglycemia and glucagon stimulation resulted in deficient GH release to a maximum of 1.4 µg/liter (normal, >7 µg/liter). He also had partial PRL deficiency with a normal basal PRL (5.4 µg/liter), but no increase in PRL response to TRH stimulation. Although he initially had normal thyroid function, he developed secondary hypothyroidism over the first 2 yr of life, as evidenced by a low serum T4 level of 39 nmol/liter (normal 58–193) and a blunted response of TSH to TRH (<1.7 mU/liter, below the limits of detection of the assay at that time) at 28 months of age. An A-to-G mutation in codon 216 of the Pit-1 gene has been identified in one-half of independent clones sequenced (data not shown), resulting in an amino acid change from a lysine to a glutamic acid in one allele (K216E). The rest of the Pit-1 gene was sequenced, and no other mutation was identified, indicating that this patient is not a compound heterozygote. The parents’ DNA could not be obtained, but they were of normal height, suggesting that this is most likely a sporadic mutation. Codon 216 encodes the third amino acid in a phosphorylation consensus sequence of the Pit-1 gene specified by the amino acid sequence (K/R)4RT(S/T)I (29). This region is completely conserved among the homeodomains of other POU domain proteins. The molecular properties of the K216E Pit-1 mutation were evaluated in this study.

Superactivation of the Mutant Pit-1 Protein on the GH and PRL Genes
The effect of the K216E Pit-1 mutant on the expression of the GH and PRL genes was investigated in CV-1 and JEG-3 cells (Pit-1 deficient). The GH and PRL genes were chosen for these studies because their responses to Pit-1 have been well characterized. A construct containing 236 bp of rat (r) GH or 1.0 kb of bovine (b) PRL 5'-flanking DNA was fused to the luciferase reporter gene. The proximal GH promoter contains both GH1 and GH2 sites (Pit-1 binding sites) and the intervening Z-box sequence (10). The human GH promoter is similar to the rat GH promoter and is virtually identical to the rat GH gene in the GH1-Z box-GH2 region. The rat Pit-1 cDNA was chosen because rat Pit-1 and its isoforms are much better characterized than human Pit-1, and rat and human Pit-1 are virtually identical at the amino acid level.

Figure 1Go illustrates the effect of wt Pit-1 and the human mutation of Pit-1, K216E, on GH and PRL gene activation. Wild-type Pit-1 activated the GH promoter 1.5-fold in both CV-1 and JEG-3 cells (Fig. 1AGo). Interestingly, the K216E mutant Pit-1 was a better stimulator of the GH promoter than wt in both CV-1 and JEG-3 cells (3.7- and 2.5-fold, respectively). The same pattern of stimulation, but to a much greater extent, was seen for the proximal PRL promoter in JEG-3 cells. Wild-type Pit-1 stimulated the proximal PRL promoter 25-fold, while the K216E mutant Pit-1 stimulated it 41-fold (Fig. 1BGo). Relative activation of the proximal PRL promoter in CV-1 cells was minimal compared with empty vector (EV) (data not shown), suggesting that other factors not found in CV-1 cells may be important in PRL gene activation. Western blot analysis (Fig. 1CGo) confirmed equivalent expression of wt and mutant K216E Pit-1 in the transfection system. Thus, the differences in activation cannot be explained by differential levels of protein expression. The increased activation of pituitary genes by the mutant Pit-1 leaves the mechanism of CPHD yet unexplained.



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Figure 1. Activation of the GH and PRL Promoters in Pit-1-Deficient Cell Lines (CV-1 and JEG-3)

A pSG5 wt or mutant K216E Pit-1 expression construct or pSG5 EV (0.5 µg in CV-1, 0.3 µg in JEG-3) was cotransfected with a 236-bp rGH luciferase reporter construct (3 µg) (panel A) or a 1.0-kb bPRL luciferase reporter construct (3 µg) (panel B). Each experiment was performed in triplicate. Data are the mean ± SE stimulation of the reporter construct after correction for transfection efficacy as monitored by pTKGH. For the rGH promoter, P < 0.01 when EV is compared with wt, and p<0.005 when K216E mutant is compared with wt; for the bPRL promoter, P < 0.005 when EV is compared with wt, and P < 0.005 when K216E mutant is compared with wt, using Student’s unpaired t test. C, Western blot analysis of the cellular extract. Equivalent amounts of wt and K216E mutant Pit-1 protein of the expected 31- and 33-kDa size isoforms were detected with a Pit-1 monoclonal antibody.

 
DNA-Binding Properties of the Mutant Pit-1 Protein
We next determined the DNA-binding properties of the K216E mutant Pit-1 molecule, since an alteration in DNA binding might explain the functional changes that were observed above. For these studies, wt and K216E mutant rat Pit-1 were expressed in Escherichia coli using a glutathione S-transferase (GST) gene fusion system. GST is synthesized as a 29-kDa protein, while both wt and mutant Pit-1 GST fusion proteins are synthesized as approximately 60-kDa proteins in equal amounts (Fig. 2AGo).



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Figure 2. DNA Binding Properties of WT and Mutant Pit-1 on the PRL-1P-Binding Site

A, An SDS-PAGE analysis after purification of the GST-Pit-1 fusion proteins revealed the expected 29-kDa GST protein and approximately equal amounts of the wt and mutant constructs. B, Gel-mobility shift analysis of the PRL response element utilizing purified Pit-1 proteins from E. coli. Equal amounts of the GST and Pit-1 proteins were used, the last lane containing GST protein only. DNA fragment used: PRL-1P, -60 TTATATATATATTCATGAA -42. The longer arrows denote specific protein-DNA complexes; the solid arrow may represent a monomer, and the dashed arrow may represent a dimer. The shorter arrow denotes the supershifted complex with the addition of Pit-1 antiserum (Pit-1 AS). The * denotes a nonspecific complex also observed with GST protein alone.

 
The DNA-binding properties of wt and K216E-purified bacterial recombinant Pit-1 were evaluated in a gel mobility shift assay using a high-affinity PRL binding site (PRL-1P). As shown in Fig. 2BGo, recombinant Pit-1 formed two distinct complexes with the PRL probe, consistent with monomeric and dimeric species. These complexes were specifically shifted with Pit-1 antisera, but not c-Jun antisera, confirming their identity. Protein from E. coli containing GST alone did not result in formation of a band. The K216E mutant protein had increased binding to the PRL site relative to wt Pit-1. An increase in DNA binding of the mutant Pit-1 protein may explain the increased transactivation properties on the GH and PRL promoters.

The K216E mutation is found in the N-terminal basic region of the POU homeodomain. Crystallographic structure of the POU homeodomain indicates that the K216E mutation lies within the spacer region between the POU-specific and POU-homeo domains (30) and does not contact DNA (31). Hence, the gel mobility shift assay confirms that codon 216 is not likely to contact the Pit-1-binding site. However, this codon may be important in interactions between Pit-1 and other proteins.

Defective RA Signaling on the Pit-1 Gene Enhancer
These data did not clarify how the K216E mutation caused CPHD, since K216E appeared to be a superactive stimulator of GH and PRL gene expression. However, recent data suggest that an important additional role for Pit-1 involves its interaction with the nuclear hormone superfamily of receptors (13, 14, 15, 16). During development, Pit-1 gene activation requires RA induction of a Pit-1-dependent Pit-1-autoregulatory feedback loop (16). Since Pit-1 functionally interacts with the RAR on the Pit-1 gene distal enhancer, defective interaction with the K216E mutant and this receptor could result in abnormal regulation of the Pit-1 gene. A lack of Pit-1 induction would then result in a cascade of events leading to disruption of pituitary gene expression. We tested, therefore, whether this mutation would alter RA signaling on the Pit-1 gene distal enhancer.

Figure 3Go is the functional assessment of this enhancer in CV-1 cells (Pit-1 deficient). pSG5 expression vectors containing either RAR-{alpha}, and wt rat Pit-1 or the K216E mutation in rat Pit-1 were cotransfected with two copies of the Pit-1 gene distal enhancer (-10.7 to -10.2 kb) fused upstream of TK109Luc. Cotransfection of the RAR with an EV expression vector yielded a minimal RA response of 1.9-fold (seventh set of bars). In contrast, wt Pit-1 and RAR cotransfection mediated a 36.0-fold response by RA (first set of bars), which confirmed a previous report showing a Pit-1 dependence for the RA response on this element (16). Cotransfection of RAR and the K216E mutant, however, resulted in a minimal RA response of 3.4-fold (sixth set of bars).



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Figure 3. Effect of a Pit-1 Mutation on RA Regulation of the Pit-1 Gene Distal Enhancer

Two copies of the mouse Pit-1 enhancer were inserted upstream of TK109Luc. This construct (0.5 µg) was cotransfected with a pSG5 RAR-{alpha} expression vector (0.1 µg) in CV-1 cells and varying amounts of pSG5 wt and/or mutant K216E Pit-1 expression construct or pSG5 wt Pit-1 and/or EV expression construct to a total of 0.5 µg. Stimulation was performed with 10-6 M 9-cis-RA for 24 h. Each experiment was performed in triplicate. Data are expressed as the mean ± SE fold activation of the Pit-1 distal enhancer reporter construct relative to EV, after correction for transfection efficacy as monitored by pTKGH. After RA stimulation, P < 0.01 when comparing 0.25 µg K216E mutant plus 0.25 µg EV with 0.5 µg wt, and P < 0.005 when comparing 0.1 µg K216E mutant plus 0.4 µg EV or 0.5 µg K216E mutant with 0.5 µg wt, using the unpaired Student t test. However, P = NS when comparing all concentrations of wt plus EV with 0.5 µg wt.

 
To determine whether cotransfection of increasing amounts of K216E mutant vs. wt Pit-1 resulted in a progressive decrease in RA induction, varying amounts of mutant Pit-1 were cotransfected with wt Pit-1 and RAR expression vectors, and the degree of RA induction was determined (Fig. 3Go). When total Pit-1 was kept constant at both a 1:1 and 4:1 mutant to wt plasmid DNA ratio (second and fourth set of bars, respectively), there was decreased RA induction of the Pit-1 gene distal enhancer element relative to wt Pit-1, to a level of 19.8- and 11.6-fold, respectively. To control for the decreasing amount of wt Pit-1 used, total DNA was also kept constant by the addition of EV (third and fifth set of bars). There was not a significant change in RA induction of the Pit-1 gene distal enhancer when the mutant K216E Pit-1 was not present, indicating that the decreased responsiveness was not due to decreased amount of functional wt Pit-1. These data indicate that this Pit-1 mutation is functionally defective and blocks wt Pit-1 in mediating RA induction of the Pit-1 gene through its distal enhancer element, with a 1:1 wt to mutant Pit-1 ratio resulting in a decrease in activation of the Pit-1 distal enhancer by approximately 50%.

Aberrant Physical Interaction between RAR and the Pit-1 Mutant on the Pit-1 Gene Distal Enhancer
To test for structural interactions between Pit-1 and RAR, gel mobility shift analysis with a radiolabeled fragment of the Pit-1-dependent RARE (PRARE) was performed. Figure 4Go is the gel shift assay with the PRARE element. RAR-{alpha} alone did not form a specific protein-DNA complex either alone or in combination with RA. wt And K216E mutant Pit-1 protein alone formed a protein-DNA complex with the probe, which was further shifted in mobility by Pit-1 antisera (Pit-1 AS), indicating that Pit-1 was present in the complex. However, the supershifted wt and mutant bands differ, suggesting that the conformation of the K216E mutant on this element could differ from wt Pit-1. When both wt Pit-1 and RAR were present in the binding reaction, a slightly larger complex was formed (long arrow), which was shifted in mobility (short arrow) by RAR-{alpha} antisera (RAR AS), indicating that wt Pit-1 and RAR bind cooperatively on the Pit-1 gene distal enhancer. The light intensity of the band is consistent with the weak cooperative binding interactions between RAR and Pit-1 on the PRARE (16). This effect was not seen with the K216E mutant Pit-1, indicating that there is decreased binding cooperativity between the mutant Pit-1 and RAR on the Pit-1 gene distal enhancer.



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Figure 4. Gel Mobility Shift Analysis of the PRARE Utilizing Purified Pit-1 Proteins from E. coli and in Vitro Translated RAR-{alpha}

One microgram of recombinant GST protein or wt or mutant K216E Pit-1 and/or 4 µl of in vitro translated RAR-{alpha} or unprogrammed reticulocyte lysate (UP) was exposed to a radiolabeled PRARE DNA fragment. The bracket denotes the supershifted Pit-1 complex with the addition of Pit-1 antiserum (Pit-1 AS). When both wt Pit-1 and RAR were present in the binding reaction, a slightly larger complex was formed (long arrow) which was shifted in mobility (short arrow) by RAR-{alpha} antisera (RAR AS) indicating that wt Pit-1 and RAR bind cooperatively on the Pit-1 gene distal enhancer.

 
Effect of the Mutant Pit-1 Molecule on Pit-1 mRNA in Vivo
To determine whether the defect of the mutant Pit-1 in mediating RA induction of the Pit-1 gene was significant in vivo, Northern hybridization analysis was performed utilizing mRNA obtained from a Pit-1-expressing cell line (GH3). Wild-type or mutant K216E Pit-1 or EV was transfected into the GH3 cell line, and Northern hybridization was performed with a rat Pit-1 cDNA probe. Figure 5aGo shows the detection of two mRNA transcripts of the expected 2.6-kb and 1.2-kb sizes (32). Figure 5bGo is the densitometric analysis of this Northern blot after correction for ß-actin levels. After transfection of wt Pit-1, RA stimulation led to an increase in Pit-1 mRNA, comparable to the level seen after RA stimulation after transfection of EV. This result was expected, since the GH3 cell line expresses endogenous Pit-1 at high levels. However, there was no increase in Pit-1 mRNA after RA stimulation when the mutant Pit-1 was transfected. Thus, the mutant K216E Pit-1 interferes with RA stimulation of the endogenous Pit-1 gene in GH3 cells.



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Figure 5. Northern Hybridization Analysis of Pit-1 mRNA Expression in GH3 Cells (Pit-1 Sufficient) after Transfection with a pSG5 wt or Mutant K216E Pit-1 Expression Construct or pSG5 EV (8 µg)

Stimulation was performed with 10-6 M all-trans-RA for 24 h. mRNA (0.5 and 1.5 µg) was used for each sample. A, mRNA transcripts of 2.6 and 1.2 kb were detected. B, Densitometric analysis of the Pit-1 mRNA. Data are expressed as Pit-1 mRNA, divided by the densitometric analysis of ß-actin in each lane to correct for loading, and expressed relative to wt Pit-1.

 
Impaired RA Signaling Leads to Defective Activation of Pit-1 Target Genes
To ascertain whether the defect in RA induction of the Pit-1 gene affected pituitary hormone gene expression, a construct containing two copies of the Pit-1 gene distal enhancer, fused upstream of the human Pit-1 promoter, linked to either wt or mutant K216E rat Pit-1 cDNA was made (Fig. 6aGo). A third vector containing no Pit-1 cDNA was also constructed. These vectors were cotransfected with a pSG5 expression vector containing the RAR-{alpha} receptor and a luciferase reporter construct containing 236 bp of the rGH promoter in CV-1 cells. Figure 6bGo shows that relative to EV, the wt construct activated the GH promoter after RA stimulation, while the K216E mutant construct was completely defective. Since the K216E mutation is found on only one allele, the wt and mutant constructs were cotransfected in a 1:1 ratio, keeping total amount constant (Fig. 6cGo). RA induction of the GH promoter was blocked, indicating that the K216E mutant acted as a dominant inhibitor of wt Pit-1 in this transfection system. To confirm that this decrease was not due to using half the amount of wt construct, the wt construct and EV construct were also cotransfected in a 1:1 ratio, keeping the total amount constant. There was RA induction of the GH promoter similar to that seen with the wt construct alone. Thus, RA induction of the Pit-1 gene distal enhancer leads to defective GH promoter activation when the K216E mutation is present, and wt Pit-1 function is inhibited as well.



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Figure 6. Activation of the GH Promoter after RA Regulation Stimulation of the Pit-1 Gene Distal Enhancer in CV-1 Cells

A, Two copies of the mouse Pit-1 gene enhancer were fused to 991 bp of the human Pit-1 promoter placed upstream of wt or mutant K216E rat Pit-1 cDNA and an SV40 polyA site in the PGEM9z vector (PRARE-Pit-1). B, This construct (1.5 µg) was cotransfected with a pSG5RAR-{alpha} expression vector (0.1 µg) in CV-1 cells and 236 bp of the rGH promoter fused to luciferase (3 µg). Stimulation was performed with 10-6 M all-trans-RA for 24 h. Each experiment was performed in triplicate. Data are standardized to EV and are the mean ± SE stimulation of the GH reporter construct after correction for transfection efficacy as monitored by pTKGH. After RA stimulation, P < 0.01 when EV is compared with wt, and P < 0.005 when K216E mutant is compared with wt using the unpaired Student t test; or using varying amounts of the constructs to a total of 1.5 µg (panel C). After RA stimulation, P = NS when comparing 0.75 µg PRARE-wt plus 0.75 µg PRARE-EV with 1.5 µg wt-PRARE using Student’s unpaired t test. However, P < 0.005 when comparing 0.75 µg PRARE-K216E mutant plus 0.75 µg PRARE-EV with 1.5 µg wt-PRARE.

 
Molecular Mechanisms Causing CPHD
Pit-1 gene mutations are found sporadically or are inherited as either autosomal-recessive or dominant traits (18, 19, 20, 21, 22, 23, 24, 25). Autosomal-recessive mutations usually disrupt Pit-1 DNA binding leading to a lack of gene activation in the pituitary. Other mutations, present on only one allele, are thought to dominantly inhibit the function of wt Pit-1 and also lead to CPHD. We have previously described one mutation (R271W) with these properties in two different patients (19, 22). In the present report, we have characterized the mechanism of transrepression by a new mutation, K216E. This mutation is found in the 5'-region of the POU homeodomain in an area that does not appear to be important for DNA binding to target pituitary genes (31). Indeed, structural analysis indicated that the mutant Pit-1 does not appear to decrease DNA binding. Since the mutant Pit-1 does not function on certain elements such as the Pit-1 distal enhancer, this region may be important for protein-protein interactions between Pit-1 and other transcription factors.

Initial studies indicated that the K216E mutant acted as a superactivator of GH and PRL gene expression, demonstrating that the K216E mutant must cause CPHD through yet another mechanism. Pit-1 is known to interact with the steroid/thyroid hormone superfamily of nuclear receptors and modify hormonal regulation of certain target genes. Pit-1 is required for estrogen induction of the PRL gene via the distal PRL enhancer and RA induction of the Pit-1 gene during development via the Pit-1 gene distal enhancer. We determined that the K216E mutation was unable to support RA induction of the Pit-1 gene distal enhancer either alone or in combination with wt Pit-1. The mutant Pit-1 was unable to interact with the RAR, and RA was unable to stimulate Pit-1 expression in vivo when the mutant K216E Pit-1 was present. Furthermore, this mutant Pit-1 resulted in defective Pit-1-dependent RA induction of the GH promoter. Since the patient has one normal and one mutant allele, the net result may be a significant reduction in RA activation of the Pit-1 gene during a crucial period of development. Our data indicate that in the patient, reduction in Pit-1 gene activation leads to decreased Pit-1 mRNA levels and presumably decreased Pit-1 protein levels. The final result is decreased activation of target genes, such as GH (model, Fig. 7Go). We suggest that the ability to selectively impair interaction with the superfamily of nuclear hormone receptors is a new molecular mechanism responsible for CPHD caused by certain Pit-1 gene mutations.



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Figure 7. Model of Selective Impairment of the Interaction of Pit-1 with the RAR as a Molecular Mechanism Responsible for CPHD

Pit-1 is required for RA induction of the Pit-1 gene during development via the Pit-1 gene distal enhancer. The K216E mutant is unable to support RA induction of the Pit-1 gene distal enhancer, either alone or in combination with wt Pit-1, resulting in decreased Pit-1 levels. The lack of Pit-1 leads to decreased activation of target genes.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Wild-type and mutant K216E rat Pit-1 cDNA, confirmed by DNA sequencing, were cloned into the EcoRI/BamHI sites of the SV40 viral expression vector, pSG5. Rat GH promoter (236 bp) or bovine PRL promoter (1.0 kb) was inserted upstream of the luciferase reporter gene in pSVOAL{Delta}5'. A 500-bp mouse Pit-1 gene distal enhancer (-10.7 to -10.2 kb, Pit-en) was obtained from a PCR of mouse genomic DNA and subjected to DNA sequencing. Two copies of this fragment were cloned as an Asp 718 fragment upstream of TK109 luciferase. Two copies of the mouse Pit-1 gene enhancer were fused to 991 bp of the human Pit-1 promoter placed upstream of wt or mutant K216E rat Pit-1 cDNA and an SV40 polyA site in the pGEM9z vector (PRARE-Pit-1). The human RAR-{alpha} was also inserted into the pSG5 expression vector (kind gift of B. Neel, Beth Israel Hospital, Boston, MA).

Transfections
Various concentrations of pSG5 Pit-1 expression constructs (total 0.5 µg per well in CV-1 cells, 0.3 µg per well in JEG-3 cells) were cotransfected with a rGH promoter (3 µg per well), bPRL promoter (3 µg per well), or Pit-1 distal enhancer (0.5 µg per well) luciferase reporter construct into CV-1 or JEG-3 cells (both are Pit-1 deficient) in triplicate using a calcium-phosphate precipitation technique (Specialty Media, Inc., Lavallette, NJ) in six-well tissue culture plates, and luciferase activity in relative light units was measured at 36 h. Various concentrations of PRARE-Pit-1 expression constructs (total 1.5 µg per well) were cotransfected with a rGH promoter luciferase reporter construct (3 µg per well) in CV-1 cells. Transfection efficiency was monitored by cotransfecting 0.4 µg per well of a human GH expression construct (pTKGH) and measuring GH secretion in the media by chemiluminescent assay (Nichols Institute, San Juan Capistrano, CA). The ability of mutant Pit-1 to interfere with the function of wt Pit-1 was assessed by cotransfecting either EV or K216E mutant Pit-1 with wt Pit-1 expression vector. In the Pit-1 enhancer experiments, 0.1 µg of pSG5 RAR-{alpha} was cotransfected per well; and 10-6 M 9-cis-RA (Hoffman-Roche, Nutley, NJ) or 10-6 M all-trans-RA (Sigma Pharmaceutical Co., St. Louis, MO) was added to phenol-free Dulbecco’s modified essential media with glutamine for 24 h before assay.

Western Blot Analysis
After transient transfection in CV-1 cells as above, proteins were extracted and Western blot analysis was performed according to published protocols (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and Amersham Life Science, Arlington Heights, IL, respectively). A Pit-1 monoclonal antibody (Pit-1 AS) (Transduction Laboratories, Lexington, KY) was used for detection.

Gel-Shift Analysis
Gel-shift analysis was performed with recombinant Pit-1 proteins and 32P-labeled DNA fragments. For testing Pit-1 binding, a high-affinity Pit-1 DNA-binding site from the bovine PRL gene (-60 to -42) was used: -60 TTATATATATATTCATGAA -42. For testing of Pit-1 and RAR binding to the Pit-1 gene distal enhancer, the composite Pit-1-RARE was used: 5'-TAATATGTGCTCAAAGTTCAGGTATGAATATAA-ATG-3' (RAR half-sites underlined, a half-site for Pit-1 binding site in bold). For these studies, RAR-{alpha} was synthesized in a coupled transcription/translation reaction (Promega Biotech, Madison WI), and 1 µg of bacterial recombinant purified Pit-1 was used. 9-cis RA (10-6 µM) (Hoffman-Roche) was added to reactions containing RAR.

Recombinant wt and mutant K216E Pit-1 were synthesized in bacteria by inserting the corresponding cDNAs into the pGEX-4T-2 vector. The pGEX-4T-2 vector, which contains GST, is induced to express Pit-1 in an E. coli strain (JM109) by 0.5 M isopropyl-ß-D-thiogalactopyranoside treatment. Recombinant Pit-1 fused to GST was purified by affinity chromatography using glutathione Sepharose 4B (Pharmacia Biotechnology, Uppsala, Sweden) using manufacturer’s instructions. As a negative control in these experiments, GST protein was used. Protein concentrations were measured in a protein assay (Bio-Rad, Hercules, CA).

The Pit-1 complexes were supershifted with a Pit-1 monoclonal antibody (Pit-1 AS) (Transduction Laboratories). As a control, c-Jun polyclonal antibody (C-Jun AS) (Santa Cruz Biotechnology, Inc.) was used. The RAR complexes were supershifted with an RAR-{alpha} polyclonal antibody (Santa Cruz Biotechnology, Inc.).

Northern Hybridization Analysis
pSG5 wt or mutant Pit-1 or EV expression construct (8 µg) was transfected into GH3 cells (Pit-1 deficient) using Lipofectamine Plus (GIBCO BRL, Gaithersburg, MD) in 175-cm2 tissue culture flasks. mRNA was isolated using a Guanidine Direct mRNA purification kit (CPG, Inc., Lincoln Park, NJ) using manufacturer’s instructions. mRNA (0.5 µg and 1.5 µg) was run on a 1.4% gel, and mRNA was transferred to Gene Screen Plus nylon membrane (NEN Life Science Products, Boston, MA). The membrane was hybridized with a wt rat Pit-1 cDNA probe prepared by random primed DNA labeling (Boehringer Mannheim, Indianapolis, IN) and then washed and probed with ß-actin (provided by M. Zakaria, Children’s Hospital, Boston, MA). Densitometry was performed using Adobe Photoshop 4.0 (Adobe Systems Corp., San Jose, CA) and NIH Image 1.6 (Scion Corp., Frederick, MD).


    ACKNOWLEDGMENTS
 
The authors wish to thank Yukiko Hashimoto, Rebecca Marier, and Diane Stafford for technical assistance. The patient was identified using a computerized database at Children’s Hospital, Boston (33). RAR-{alpha} was the kind gift of B. Neel (Beth Israel Hospital, Boston, MA), and ß-actin was the kind gift of M. Zakaria (Children’s Hospital, Boston, MA).


    FOOTNOTES
 
Address requests for reprints to: Laurie E. Cohen, M.D., Children’s Hospital, Division of Endocrinology, Enders 4, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail:cohen l{at}a1.tch.harvard.edu

This work was supported by grants from the NIH (L.E.C., S.R.) the Charles H. Hood Foundation (L.E.C.), and the Genentech Foundation for Growth and Development (S.R.).

Received for publication March 17, 1998. Revision received October 5, 1998. Accepted for publication November 30, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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