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Endocrinology Vol. 142, No. 7 2937-2945
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification, Isolation, and Cloning of Growth Hormone (GH)-Inducible Interscapular Brown Adipose Complementary Deoxyribonucleic Acid from GH Antagonist Mice1

Yuesheng Li2, Bruce Kelder and John J. Kopchick

Edison Biotechnology Institute (Y.L., B.K., J.J.K.), Molecular and Cellular Biology Program, Department of Biological Sciences (Y.L.), and Department of Biomedical Sciences (J.J.K.), Ohio University College of Osteopathic Medicine, Athens, Ohio 45701

Address all correspondence and requests for reprints to: John J. Kopchick, Ph.D., Konneker 206A, Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701-2979. E-mail: kopchick{at}ohiou.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In a dwarf mouse line that expresses a GH antagonist, we found that the interscapular brown adipose tissue (iBAT) mass is significantly greater than that in nontransgenic littermates. We proposed that gene expression in iBAT may be up- or down-regulated by GH. To identify these genes, we employed the PCR-select subtraction approach to construct subtractive libraries from iBAT total RNAs. We have generated forward and reverse subtractive libraries. Clones were screened by differential hybridization and identified by BLAST similarity to expressed sequence tags and complementary DNA sequences. Four novel expressed sequence tags were isolated from the reverse subtractive library. Of them, clone 42, was further analyzed. It encodes a 2475-bp messenger RNA with an open reading frame of 346 amino acids. Northern blot analysis demonstrated two RNA isoforms (2.5 and 1.3 kb) in various tissues. Differential expression of both isoforms was verified in GH antagonist and nontransgenic mouse iBAT. BLAST searches suggested that clone 42 is highly homologous to a gene found in a human female fetal brain and a related gene found in a human pituitary tumor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHs MUST BIND to the GH receptor (GHR) to participate in the regulation of normal animal growth and development. The binding mechanism has been extensively studied in our laboratory and others (1, 2, 3, 4, 5, 6, 7, 8, 9). A substitution of Arg or Lys for Gly in the third {alpha}-helix of human (h) GH generates a GH antagonist (GHA) and leads to dwarf phenotype in vivo (1, 2, 3, 4, 5, 6). Similarly, disruption of exon 4 of the GHR gene that encodes a portion of the ligand-binding region of the extracellular domain in the receptor results in a dwarf mouse. This animal is referred to the GHR/binding protein (BP) gene disrupted, GHR/binding protein (BP) knockout (KO), or Laron mouse (10).

The 2.8-Å crystal structure of hGHBP and hGH has been solved and confirms that GH can exist in a one-ligand/two-receptor complex or dimer (9). Dimerization of GHRs is thought to be responsible for transduction of GH-mediated signals (11, 12) through a series of phosphorylation events to various intermediate signal mediators, including Janus kinase-2 (13) and signal transducer and activator of transcription family members (14, 15, 16, 17, 18, 19). However, GH-specific regulation of targeted gene expression remains unclear.

It is well known that GH has pleiotropic biological effects on a variety of tissues (20). One of most pronounced functions is GH’s lipolytic effect that occurs in adipose tissue. In the GH-fat cycle model (21), GH is thought to reduce and redistribute body fat. On the other hand, obesity can be characterized by reduced GH output. In this regard, GH secretion patterns are markedly altered in obese individuals. There is an overall 4-fold reduction in the GH secretion in obese men compared with normal weight men (22). GH deficiency in children leads to an obese state (23, 24). Exogenous GH reduces obesity in individuals with GH deficiency by decreasing average cell and tissue size and lipid content of sc fat (25, 26).

GH has been shown to increase the activity of hormone-sensitive lipases (27, 28) and reduce the activity of lipoprotein lipase (29, 30). Vernon et al. (27) reported that the increased proportion of hormone-sensitive lipases by GH is associated with fat droplets during the rat lactation cycle. In clinical studies of obese women, injection of exogenous hGH decreases body fat (31), reduces adipose lipoprotein lipase activity, and enhances the plasma level of FFA (29).

In a previous report, Knapp et al. found that food intake was proportional to body size in GHA mice and nontransgenic (NT) littermates during the postweaning period of rapid growth (32). The feed efficiencies (gain/feed) and growth rates of GHA mice were similar to those of NT littermates. GHA mice had increased body fat and decreased body protein percentages compared to NT littermates (P < 0.05) (32). We observed that GHA mice tend to catch up with NT littermates in body weight, but not in body length, over time (Li, Y., and J. J. Kopchick, manuscript submitted). Thus, normalizing body weight to body length, the aged GHA mice possess a greater body mass index (BMI) value than that of NT littermates. The higher BMI with an unchanged feed efficiency implies a shift in energy homeostasis in these GHA obese mice. The body composition was also altered in those transgenic mice (32).

Brown adipose tissue (BAT) is located in the interscapular region in rodents and is a site of heat production or nonshivering thermogenesis. BAT has higher metabolic activity per cell than WAT (33). We have found that the interscapular BAT (iBAT) mass in the GHA mice was significantly greater than that found in nontransgenic (NT) littermates (Li, Y., and J. J. Kopchick, manuscript submitted). Moreover, this enlargement is not proportional to body size and is also observed in GHR/BP KO mice. GHR and GHRBP (GHR/BP) messenger RNAs (mRNAs) were detected in normal mouse iBAT. BAT also showed limited GH binding, suggesting that mouse iBAT is responsive to GH. As GH-mediated signaling negatively regulates the expression of the mouse UCP1 gene that is specific to iBAT, we hypothesized that GH may regulate gene expression important for the phenotypic changes in GHA mice. In this report we describe the identification, isolation, and cloning of a GH-inducible iBAT complementary DNA (cDNA), clone 42, from GHA mice using a PCR-select subtraction approach.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
GHA and GHR/BP KO mice have been described previously (1, 6, 10). Serum GH antagonist (GHA) levels ranged from 0.4–10 µg/ml in GHA mice (1, 6), whereas GHR was undetectable in GHR/BP KO mice (10). All mice were fed ad libitum with Purina rodent chow 5004 (Ralston Purina Co., St. Louis, MO) and housed under standard environmental conditions. All procedures were approved by the Ohio University institutional animal care and use committee.

Total RNA preparations
Mice were killed after weighing at 10 or 52 weeks of age. The interscapular adipose tissues (iAT) depot, which consists of yellowish white interscapular WAT (iWAT) peripherally and reddish brown interscapular BAT (iBAT) centrally, was isolated. Subcutaneous WAT (scWAT), gonadal WAT (gWAT) that includes epididymal WAT (eWAT) and ovarian WAT (oWAT), brain, liver, kidney, spleen, heart, muscle, lung, intestine, skin, and testis or ovary also were immediately dissected and weighed. All tissues were placed in 10 vol cold RNA STAT-60 reagent (Tel-Test, Friendswood, TX), and homogenized on ice as described in the manufacturer’s suggested protocol.

PCR probe labeling
For PCR probe labeling, the sets of primers described below were used and are located in the coding region of the cDNA. The numbers in parentheses are the expected size (base pairs) of PCR products: ß-actin, 5'-TGTCAGGTCTTCTTAACCTTGG-3' and 5'-CCACGAATCCCGGTCAAACTAATGT-3' (540 bp); UCP1, 5'-GCCAGGCTTCCAGTACCATT-3' and 5'-GGTACTGTCCTGGCAGAGAGTT-3' (605 bp); C42 open reading frame (ORF), 5'-CTAATGAGATTGGAGCTATC-3' and 5'-GCA TAGTTGCAACTCGCA-3' (699 bp); and C42 3'-end, 5'-ATACAGAGAATCTCTGCTGG-3' and 5'-CGATAGCTCCAATCTCATTAG-3' (549 bp). Mouse ß-actin, mouse UCP1 (34), and pBluescript SK+ C42 plasmids were used to prepare PCR-digoxigenin (Dig)-labeled probes and PCR-32P-labeled probes.

For PCR-Dig probe labeling, a probe synthesis mix containing Dig-11-deoxy (d)-UCP was mixed with PCR buffer, 0.2 pmol antisense strand primer, 0.1 ng PCR-amplified DNA fragment, and 0.4 µl Advantage-HF 2 Polymerase Mix in total volume of 20 µl following the instructions in the PCR DIG Probe Synthesis Kit (Roche, Indianapolis, IN).

For preparation of the PCR-32P-labeling probe, 20 µCi each of [{alpha}-32P]dCTP and [{alpha}-32P]dTTP were mixed with 20 nmol each of dATP and dGTP, PCR buffer, 0.2 pmol antisense strand primer, 0.1 ng PCR-amplified DNA fragment, and 0.4 µl Advantage-HF 2 Polymerase Mix in total volume of 20 µl following the protocols in the StripAble PCR probe Synthesis Kit (Ambion, Inc., Austin, TX). Unincorporated radionucleotides were removed by gel filtration using STE SELECT-D G-25 columns (Eppendorf, 5 Prime->3 Prime, Boulder, CO). Probe-specific activities were determined using a MultiPurpose scintillation counter (LS 6500, Beckman Coulter, Inc., Palo Alto, CA).

Northern analyses
Total RNA samples were resolved by 1% formaldehyde/agarose gel electrophoresis, transferred overnight to a positively charged nylon membrane (Roche), cross-linked to the membrane using a UV Stratalinker oven (Stratagene, La Jolla, CA), prehybridized with either Dig Easy Hyb solution (Roche) or freshly made prehybridization buffer [6 x SSPE (0.15 M NaCl, 10 mM NaH2 PO4, 1 mM EDTA; pH 7.4), 5 x Denhardt’s reagent, 0.5% SDS, 1.0 mg/ml salmon sperm DNA, and 50% deionized formamide] at 50 C for 1 h, and then hybridized with either 10 ng/ml PCR Dig-labeled or 1 x 106 cpm/ml PCR 32P-labeled probes in a Micro Hybridization Incubator (model 2000, Robbins Scientific, Mountain View, CA) at 50 C for 16 h. Washing procedures and detection of Dig-labeled nucleic acids were described in the Genius System User’s Guide (Roche). For detection of 32P-labeled nucleic acids, membranes were washed once with 1 x SSC/0.1% SDS at room temperature for 20 min and three times with 0.2 x SSC/0.1% SDS at 68 C for 20 min, wrapped, exposed to Kodak Bio-Max MR film (Eastman Kodak Co., Rochester, NY), and developed in a Konica SRX-101 Medica Film Processor (Konica Corp., Wayne, NJ). For detection of 32P-labeled nucleic acids, film was exposed at -80 C. Northern blot images were scanned using an Agfa Duoscan T1200 scanner installed with Agfa fotolook PS 3.05 and Adobe Photoshop 4.0.1 software. The intensity volumes of individual signals were determined using Molecular Analyst version 2.1.2 software (Bio-Rad Laboratories, Inc., Hercules, CA). The volume of signal intensity from each sample was determined and compared with that of control samples.

Two-way subtractive library construction
One microgram each of total iBAT RNA from GHA and NT mice served as starting material to amplify iBAT cDNAs as described in the SMART PCR cDNA Synthesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The experimental tester cDNAs were ligated to adaptors and labeled as GHA or NT adaptor-ligated cDNAs, whereas the experimental driver cDNAs were unligated and labeled as GHA or NT adaptor-free cDNAs as described in the PCR-Select cDNA Subtraction Kit (CLONTECH Laboratories, Inc.). Two subtractive libraries were prepared as follows. A forward subtractive library was prepared by subtracting NT adaptor-free cDNAs from GHA adaptor-ligated cDNAs, and a reverse subtractive library was generated by subtracting GHA adaptor-free cDNAs from NT adaptor-ligated cDNAs. cDNAs from either subtractive library were further amplified in a two-step PCR reaction. Secondary PCR amplification products were cloned into a 3.9-kb PCR II vector (Invitrogen, Carlsbad, CA) as described in the TA Cloning Kit and then transformed into DH5{alpha} competent cells (Life Technologies, Inc., Grand Island, NY). In total, 320 colonies (160 from each subtractive library) were selected and screened.

Subtractive library screening
Two random primed Dig probes were prepared as subtractive library screening probes from the secondary PCR amplification products following the instructions in the DIG High Prime DNA Labeling Kit (Roche). cDNA arrays were prepared following the protocol provided with the PCR-Select Differential Screening Kit (CLONTECH Laboratories, Inc.). Briefly, 320 colonies were in situ PCR amplified and blotted onto positively charged nylon membranes (Roche ) in duplicate. Each blot contained 40 clones from the forward subtractive library and 40 clones from the reverse subtractive library. Each duplicate was hybridized with each subtractive library screening probe and processed as described for Northern blot analysis above. The ratio of intensity volume of candidate clones over background was used to identify positive clones from the subtractive library. Forty positive candidates were selected of 320 clones by differential signal intensities. Of these, 26 clones were from the forward subtractive library, and 14 were from the reverse subtractive library. DNA inserts of positive clones from both subtractive libraries were prepared with the Plasmid Midi Kit (QIAGEN, Chatsworth, CA).

DNA sequencing
The partial cDNA inserts obtained from subtractive libraries were sequenced as described in the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech, Cleveland, OH). [33P]Dideoxy-NTP nucleotides (NEN Life Science Products, Boston, MA) were used for signal detection. The full-length clone 42 cDNA was sequenced twice using both strands as described previously and/or by following the instructions with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp., PE Applied Biosystems, Foster City, CA). DNA sequences were resolved by the automated ABI PRISM 310 Genetic Analyzer and edited with the ABI PRISM EditView 1.0.1.sea software. The entire nucleotide sequence of clone 42 was determined by "walking through" the sequence starting with the SK primer (5'-CGCTCTAGAACTAGTGGATC-3') and the modified KS (5'-CCTCGAGGTCGACGGTATC-3') primer.

cDNA library construction
BAT cDNAs were synthesized as described previously. The cDNAs were ligated into the {lambda}ZAPII vector and packaged with Gigapack III Gold packaging extract as described by the manufacturer of the ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene). The integrity of iBAT cDNAs used for library construction was confirmed by PCR amplification of the ß-actin, UCP1, and GHR/BP genes and resolved by 1% agarose gel electrophoresis. Primers for ß-actin and UCP1 are as described for PCR probe labeling above, whereas the primers for GHR/BP were 5'-GCCAGGCTTCCAGTACCATT-3' and 5'-GGTACTGTCCTGGCAGAGAGTT-3'. The titer of the amplified cDNA library was approximately 4.30 x 105 plaque-forming units/µl.

cDNA library screening
cDNA Library Screening was conducted as described in the manual of ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene). Briefly, 8.60 x 105 plaque-forming units of the amplified library were screened with a Dig-labeled Clone Reverse 36 (R36) probe selected from the reverse subtractive library. Plaque lifts were performed using nylon membranes (Roche). The hybridization reaction was as described for Northern analysis above. One positive {lambda}ZAPII Vector plaque was isolated and purified by three rounds of selection and named clone 42. The pBluescript SK (pBSK) phagemid containing clone 42 was excised from this purified plaque in the SOLR strain of Escherichia coli as described in the manual (Stratagene). pBluescript SK+ C42 plasmid DNA was then prepared with the Plasmid Maxi Kit (QIAGEN, Chatsworth, CA) for further studies.

Construction of clone 42 mammalian expression vector
To detect the clone 42 protein product in transfected mammalian cells, a C-terminal histine tag was engineered into the cDNA. The C42His cDNA fragment that contains a BglII site (lower case letters) at the 5'-end of the sense strand was PCR engineered from the pBluescript SK+ C42 plasmid using a 5'-primer (5'-cgcggatccagatctaccATGTTGGCTGCAAGGCTTGTGTGTC-3') and a 3'-primer (5'-cgcggatccTCAATGGTGATGGTGATGGTGTTTCTTTCTGTTGCTTCCAGTTGCTAGCATA-3'; Fig. 1Go). This cDNA sequence includes clone 42 ORF (upper case letters) and six consecutive histidine residues (upper case letters) at the C-terminus. The C42His cDNA fragment was digested with BglII and ligated into pMET-bGH-C that had previously been digested with both BglII and PvuII (New England Biolabs, Inc., Beverly, MA). The linearized pMET-bGH-C contains a mouse metallothionein I promoter and a bovine (b) GH polyadenylation signal sequence. The ligation product was then transformed into DH5{alpha}-competent cells as described previously. A recombinant colony was isolated, amplified, and purified with the Plasmid Midi Kit (QIAGEN). Automated DNA sequencing was employed to verify the sequence of cDNA insert in pMET-bGH-C42His plasmid as described for DNA sequencing.



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  		Figure 1. Generation of clone 42 expression vector. A C42His cDNA fragment that contains a BglII site at the 5'-end of the sense strand was PCR-engineered from pBluescript SK+ C42 plasmid, digested with BglII, and ligated into pMET-bGH-C plasmid that had previously been linearized with both BglII and PvuII. The C42His cDNA sequence includes clone 42 ORF and six consecutive histidine residues at the C-terminus. The new pMET-bGH-C42His plasmid retains a mouse metallothionein I promoter and a bGH polyadenylation (bGH Poly A) signal sequence that are derived from parent vector, pMET-bGH-C.

 
Expression of clone 42 in mouse L cell line
Mouse L cells were cotransfected with pMET-bGH-C42His and pD{lambda}AT3 using calcium phosphate precipitation method as previously described (35, 36, 37). Transfected mouse L cells were cultured to 80–100% confluence, washed, scraped with 1 x cold PBS, and centrifuged in microcentrifuge tube at 500 x g at 4 C for 5 min. Each cell pellet was then incubated with lysis buffer (1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and PBS, pH 7.4) containing a protease inhibitor solution [10 µg/ml phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM sodium orthovanadate] in microcentrifuge tube on ice for approximately 20 min and centrifuged at 1500 x g at 4 C for 15 min. Protein concentrations of cell lysates were determined by protein assay (Bio-Rad Laboratories, Inc.).

Western analysis
Western blotting was performed as described in the antibody kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, mouse L cell proteins were resolved by 10% SDS-PAGE and transferred to an NC Pure Nitrocellulose Transfer and Immobilization Membrane (Schleicher & Schuell, Inc., Keene, NH). Membranes were blocked with Blotto A and serially incubated with a 1:1,000 dilution of a mouse monoclonal IgG1 directed against the His-tag (H-3, Santa Cruz Biotechnology, Inc.) and with a 1:10,000 dilution of the secondary antibody directed against mouse IgG and conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.). The ECL Western blot detection reagent (Amersham International, Little Chalfont, UK) was used for signal visualization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A differential signal of clone reverse 36 in GHA mouse iBAT
To examine the hypothesis that GH regulates genes in iBAT, two-way subtractive libraries were constructed as described above. From the reverse subtractive library, 160 colonies were isolated. Forty clones were loaded onto nylon membranes in duplicate. The first membrane was probed with the reverse subtractive library, whereas the second was probed with the forward subtractive library and served as a negative control. Fourteen of 160 clones were found to be differentially expressed (Li, Y., and J. J. Kopchick, in preparation).

The sequence identity of 14 candidates were determined by the similarity to EST and/or cDNA sequences from the database of Nonredundant GenBank CDS using the Basic Local Alignment Search Tool (BLAST) (38). 4 out of the 14 candidates did not show significant similarity to sequences present in the database, and were considered to be "novel" EST candidates.

One of the 4 EST was Clone Reverse 36 (R36) and is circled in the dot blot (Fig. 2Go). The R36 signal was detected in the reverse-probed blot, but gave no signal in the forward-probed blot. Hence, R36 was considered to be a novel EST candidate that might be negatively regulated in GHA mice. R36, as a partial cDNA, was later used to screen, isolate, and purify a cDNA named clone 42 that encodes a large ORF. Analyses of the remaining 13 clones will be reported in the separate manuscript.



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  		Figure 2. PCR-Select differential screening of a reverse subtractive library. A reverse subtractive library was constructed by subtracting GHA iBAT cDNAs from NT iBAT cDNAs as described in Materials and Methods. One hundred and sixty colonies were isolated from the reverse subtractive library. Forty clones were loaded to each duplicate blot. The first duplicate (blot A) was probed by the reverse subtractive library probes, and the second duplicate (blot B) was probed by the forward subtractive library probes. Fourteen clones demonstrated a differential signal. Four of 14 candidates were identified as novel ESTs by BLAST searches. Of them, clone reverse 36 (R36, circled) had a relatively high signal intensity volume in the sample blot (blot A), but it was undetectable in the control blot (blot B). Thus, the ratio of the signal present in blot A to that in blot B approaches infinity.

 
Molecular characteristics of clone 42
Clone 42 was isolated and purified with a Dig-labeled R36 probe by three rounds of selection. It was sequenced and found to contain 2475 bp and possessed two potential polyadenylation tail signals (AAUAAA; Fig. 3AGo), one located within nucleotides +1321 to +1326 and the other within bases +2455 to +2460. The sequence of the probe R36 was found intact at the 3'-end of clone 42 from nucleotides +1971 to +2433. Two ORFs on the sense strand and seven ORFs on the antisense strand were found. The largest ORF (ORF 1) is from nucleotides +112 through +1152, but the context of its translational start codon does not strictly comply with the Kozak’s rule (39).



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  		Figure 3. Sequence alignments of clone 42 to genes found in GenBank Database. A, cDNA sequence alignment of clone 42 to genes found in GenBank database. Clone 42 shares approximately 90% identity with the C25077 gene (1397 bp, accession no. AF131820.1) found in female human infant brain and the PTD010 gene (2364 bp, accession no. AF078863.1) found in a human pituitary tumor. The human dermal papilla-derived protein 2 gene (DERP2; accession no. AB009685) is identical to C25077. Beyond nucleotide +1348, the 3'-end of clone 42 sequence does not match any sequence from the gene database. The translational start and stop codons are shown in black boxes, whereas the polyadenylation signal is indicated in gray boxes. Gray fonts show the EST R36 sequence. Asterisks represent the continuity of the sequence alignments that include both matched and unmatched portions. A dash means skipped bases within matched sequences. B, Amino acid sequence alignment of clone 42 to proteins found in GenBank database. A signal peptide cleavage site in the 346-amino acid clone 42 polypeptide is predicted, most likely between residue Q18 and P19. The predicted protein encoded by clone 42 shares strong identity to proteins derived from C25077 and PTD010, but a relatively low similarity to CG1287 protein (accession no. AE003671.1) found in D. melanogaster.

 
The predicted ORF of clone 42 encodes a 346-residue polypeptide that possesses a potential signal peptide cleavage site, most likely between residues Q18 and P19 (Fig. 3BGo) (40). This protein is also predicted to be stable with a molecular mass of 36 kDa, a theoretical isoelectric point of 9.82, an aliphatic index of 97.05, and a grand average of hydropathicity of 0.382. These data were obtained from the output of ProtParam tool (http://www.expasy.ch/tools/protparam.html). The prediction of secondary structure for clone 42 suggests an {alpha}-helical protein that contains 38.44% {alpha}-helixes, 22.54% extended strands, 12.72% ß-turns, and 26.30% random coils using the SOPM method (41) and 51.73% {alpha}-helixes, 16.18% extended strands, 5.49% ß-turns, and 26.59% random coils using the SOPMA method (42).

The polypeptide encoded by clone 42 is also predicted to contain six to eight transmembrane regions (43) and may be similar to a cell surface protein, bacteriorhodopsin, as predicted by the 3D-PSSM Program (44). Potential O-type glycosylation sites are predicted at S186, T163, and T269 (45, 46, 47). Potential phosphorylation sites are predicted at S71, S127, S 182, T50, T54, T59, T306, T322, Y46, and Y183 (48).

Down-regulation of clone 42 in GHA and GHR/BP KO mouse iBAT
To verify the differential expression of clone 42 in mouse iBAT, RNA from 10-week-old GHA, GHR/BP KO, and NT littermates was isolated for Northern analyses. Two bands, 2.5 and 1.3 kb, were detected in iBAT using Dig-labeled C42-ORF probe (Fig. 4AGo). The levels were reduced in each of the dwarf transgenic mice compared with NT littermates. For the long isoform, the ratio of intensity volume of clone 42 to ß-actin was reduced to 35% in GHA mice and 33% in GHR/BP KO mice relative to that in NT littermates. For the short isoform, the ratio was reduced to 30% in GHA mice and 28% in GHR/BP KO mice relative to that in NT littermates.



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  		Figure 4. Northern analysis of clone 42 in adipose tissues of 10-week-old GHA and GHR/BP KO Mice. Total RNA samples were prepared from iBAT and eWAT of GHA, GHR/BP KO, and NT littermates at 10 weeks of age. A, Two isoforms, a 2.5-kb and a 1.3-kb form, were detected in all iBAT samples using the Dig-labeled C42-ORF probe, but levels were strikingly reduced in both transgenic mice compared with those in NT littermates. B, Only the 2.5-kb band was detected in iBAT and eWAT samples using the Dig-labeled C42–3'-end probe. The levels were decreased in both transgenic mice compared with those in NT littermates.

 
The Dig-labeled C42–3'-end probe detected only a 2.5-kb band in all mouse adipose tissues (Fig. 4BGo), although the signals were marginally detected in eWAT. These signals were reduced in both transgenic mouse iBAT samples. The ratio decreased to 9% in GHA mice and 50% in GHR/BP KO mice relative to that in NT littermates. Hence, both isoforms of clone 42 are down-regulated in dwarf GHA mice and GHR/BP KO mice in contrast to NT littermates. The observation of two isoforms by Northern analysis supports the possibility that two polyadenylational signals may be used in processing the clone 42 precursor mRNA.

Tissue distribution analysis of clone 42
To examine the tissue distribution of clone 42, RNA samples were prepared from both male and female mice killed at 10 weeks of age. Two bands, 2.5 and 1.3 kb, were identified in all RNAs from iBAT, scWAT, gWAT, liver, kidney, muscle, heart, spleen, intestine, brain, lung, skin, and testis or ovary (Fig. 5Go). Hybridization signal levels were more pronounced in iBAT and brain for the long isoform and were more striking in iBAT and testis for the short isoform (Fig. 5AGo). However, the Dig-labeled probe C42–3'-end only detected the long isoform across most tissues on a duplicate blot and only after a prolonged exposure (Fig. 5BGo). Signals of UCP1 were detected only in iBAT (Fig. 5CGo), which was consistent with previous reports (34). The Dig-labeled ß-actin probe was used to hybridize one duplicate blot and served as a control in this study (Fig. 5DGo). Unexpectedly, the expression pattern of ß-actin was different from tissue to tissue, specifically for muscle, heart, and testis.



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  		Figure 5. Northern analysis of clone 42 in various tissues of 10-week-old mice. Total RNAs were prepared from iBAT, scWAT, gWAT, liver, kidney, muscle, heart, spleen, intestine, brain, lung, skin, and testis or ovary of both male and female mice at 10 weeks of age. Four Northern blots were prepared and probed with different Dig-labeled probes. A, Clone 42 revealed two isoforms, a 2.5-kb and a 1.3-kb band, in all tissues with the C42-ORF probe, but levels were more pronounced in iBAT and brain for the long isoform and were more striking in iBAT and testis for the short isoform. B, Only the 2.5-kb band was detected in most tissues by the C42–3'-end probe after a prolonged exposure. C, UCP1 is only detected in iBAT using a UCP1 probe. D, ß-Actin served as the control for this study. Expression patterns of ß-actin were different from tissue to tissue when probed with the Dig-labeled ß-actin probe.

 
Regulation of clone 42 in GHA mouse iBAT as a function of age
To evaluate the relation of clone 42 to the iBAT accumulation and obese phenotypic change in GHA mice with age, iBAT and iWAT as well as eWAT were studied in GHA mice and NT littermates at 10 and 52 weeks of age. Two isoforms of clone 42 transcript were observed by Northern blotting (Fig. 6AGo). The ratio of signal intensity volume of either clone 42 isoform to 18S (Fig. 6BGo) was used to estimate the normalized levels of clone 42 transcripts in various adipose tissues.



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  		Figure 6. Northern analysis of clone 42 in adipose tissues of GHA mice at different ages. Total RNAs from iBAT, iWAT, and eWAT were examined in GHA mice and NT littermates at 10 and 52 weeks of age. A, Clone 42 transcripts were detected by the 32P-labeled C42-ORF probe and showed two isoforms after prolonged exposure. B, The 18S ribosomal RNA (18S) was used as a control. C, The ratio of signal intensity volume of either clone 42 isoform to 18S was employed to estimate the normalized levels of clone 42. Levels of both isoforms for iBAT were lower in GHA mice than in NT littermates at 10 weeks of age, but the difference diminished at 52 weeks of age (indicated by asterisk). Thus, expression levels of clone 42 may be up-regulated with age in GHA mice.

 
Compared to NT littermates, the average ratio for iWAT in GHA mice was reduced to 72% for the long isoform and 84% for the short isoform at both 10 and 52 weeks of age. In 10-week-old GHA mouse iBAT, the ratio was reduced to 53% for the long isoform and 72% for short isoform; however, these reductions diminished, because the ratio was sustained at about 96% for both isoforms in 52-week-old GHA mouse iBAT. Hence, the levels for both isoforms in iBAT were lower in GHA mice than in NT littermates at 10 weeks of age, but not at 52 weeks of age (Fig. 6CGo).

Compared to 52-week-old mice, the average ratio in iWAT was increased to 134% for both isoforms in GHA mice and NT littermates at 10 weeks of age. However, for 10-week-old GHA iBAT, the ratio was reduced to 53% for the long isoform and 77% for the short isoform, whereas in 10-week-old NT mouse iBAT, the ratio was sustained at approximately 99% for both isoforms. Thus, the levels of clone 42 mRNA may be up-regulated in GHA mouse iBAT with age, indicating a possible catch-up in expression of clone 42 during iBAT development in GHA mice (Fig. 6CGo).

In GHA mice, the average ratio for eWAT was maintained at approximately 115% compared with that in NT littermates at both 10 and 52 weeks of ages. The ratios at 10 weeks of age were reduced to 88% for the long isoform and 63% for the short isoform than that at 52 weeks of age. These data suggest that in eWAT, age-based factors may play a more important role in gene regulation of clone 42 than GH-mediated signaling.

Sequence alignments of clone 42 to genes found in GenBank catabase
Regions of sequence similarity to known genes were identified by BLAST searching (38). Many genes have been found to match clone 42 beyond the nucleotide +1794 in the sense strand, but these "hits" span approximately 100 bp. Only a few of the gene sequences exhibit similarity from the 5'-end and through the majority of clone 42. One such gene, C25077 (accession no. AF131820.1) was found in female human infant brain. Another gene, PTD010 (accession no. AF078863.1), was found in human pituitary tumor. A third gene, the human dermal papilla-derived protein 2, gene (DERP2; accession no. AB009685) is identical to C25077 at the nucleic acid level.

In DNA alignments (Fig. 3AGo), clone 42 shares approximately 90% homology with both human gene sequences, 946 of 1049 bp with C25077 and 945 of 1049 bp with PTD010. C25077 was 1397 bp long, whereas PTD010 was 2364 bp long, which corresponds to the observation of two isoforms from Northern analysis. However, beyond nucleotide 1348, the 3'-end of clone 42, the location recognized by the R36 probe, does not match any sequence from the gene database. This suggests that the 3'-end of clone 42 sequence is species specific.

In protein alignments, 12 hits have been found to match the sense strand sequence of clone 42, but only 3 of them had a significant protein homology. They are the C25077 gene product, the PTD010 gene product, and the CG1287 gene product from Drosophila melanogaster (accession no. AE003671.1). The predicted polypeptide of clone 42 shares 86% identity (298 of 346 amino acids) with the C25077, 74% identity (256 of 346 amino acids) with the PTD010, and 40% identity (139 of 346 amino acids) with the CG1287. An insertion of Phe21 in clone 42 protein causes it to be one residue longer than C25077 protein (Fig. 3BGo). No similarity has been observed between clone 42 and CG1287 at a nucleic acid level despite a relatively low homology found at the amino acid level.

Expression of clone 42 in mammalian cells
Western analysis showed that clone 42 could be expressed in mouse L cells (Fig. 7Go). The predicted ORF of clone 42 encodes a protein of 36 kDa. However, Western analysis revealed a 42-kDa band. The increased size of the protein may be due to posttranslational modification, such as glycosylation and/or phosphorylation.



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  		Figure 7. Expression of clone 42-His-tagged protein in mouse L cells. Cell lysates were resolved by 10% SDS-PAGE and immunoblotted using a mouse monoclonal antibody directed against the histidine tag. Lane 1, Mock expression in the presence of plasmid pD{lambda}AT3. Lane 2, Lysates from mouse L cells cotransfected with pMET-bGH-C42His and pD{lambda}AT3. One band was observed in the position of 42 kDa that is slightly greater than the predicted size of 36 kDa. Lane 3, Lysates from mouse L cells. Lane 4, Positive control, Par-4 (proteinase-activated receptor 4), containing a C-terminal His tag.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As previously discussed, GHA mice accumulate fat with an increased BMI as a function of age, in contrast to the NT littermates. We have also observed that from 10–52 weeks of age, the entire iAT lobes that include iBAT and iWAT became relatively larger in weight and lighter in appearance in GHA and NT mice. The moderate volumes of lipid phase were also observed from iWAT during total RNA preparation in contrast to both iBAT and eWAT (data not shown). Surprisingly, expression patterns of ß-actin varied from tissue to tissue, including iBAT, iWAT, and eWAT (Li, Y., and J. J. Kopchick, manuscript submitted). The abundance of ß-actin mRNA increased from 10 to 52 weeks of age with a relatively lower level in GHA mice in contrast to NT littermates. This in vivo observation is contradictory to the in vitro reports (49, 50). As a component of the cytoskeleton, induction of ß-actin may be regulated differently in vivo. An inverse relationship of iBAT growth to body size in the GHA and GHR/BP KO mice suggests a change in the brown adipocyte population that may contribute to the phenotypic changes in these mice.

In this study we have identified clone 42. This cDNA contains 2475 bp with two potential polyadenylational signals. At the transcriptional level, both isoforms of iBAT clone 42 are down-regulated in GHA and GHR/BP KO mice in contrast to NT littermates, but are positively regulated with age in GHA mice. Such age-related up-regulation was also observed in iWAT of GHA mice. In our separate report (Li, Y., and J. J. Kopchick, manuscript submitted), GH-mediated signaling tended to negatively regulate UCP1 gene expression in iBAT, and UCP1 signal was detected in iWAT at low levels. These data suggest that the differential gene expression may be related to the population of brown adipocytes in iBAT and iWAT during animal growth and development. The catch-up expression of clone 42 may be one example of this altered gene expression.

The predicted ORF of clone 42 encodes an {alpha}-helical polypeptide of 346 amino acids with low hydropathicity and high aliphatic index. It suggests that clone 42 protein product may be a relatively soluble and stable protein. An 18-amino acid signal peptide and six to eight transmembrane regions may direct this protein to the cell membrane. Potential posttranslational modifications include phosphorylation and type O-glycosylation. This is consistent with the observed size of the expressed protein that was 42 kDa compared with the predicted 36 kDa size. A folding model suggested a relationship of clone 42 to bacteriorhodopsin, a protein that is important for proton conductance in archaebacteria.

At both nucleic acids and amino acids levels, clone 42 is highly homologous to the C25077 gene found in female human infant brain, the PTD010 gene found in a human pituitary tumor, and the human dermal papilla-derived protein 2 gene (DERP2). In the alignment of C25077 and PTD010, a single adenine nucleotide deletion at position 1034 of PTD010 causes an ORF shift and halts translation at position 1086 instead of 1163. Thus, PTD010 encodes a 319-amino acid protein compared with 345 amino acids for C25077. Potential phosphorylation sites in clone 42 protein suggest that multiple signal sites are present, especially T306 and T322, since they are lost in the C-terminus of the PTD010 protein. This implies that the C-terminal region may be important for normal biological function and that alternation at the C-terminus may convert the novel function to an onco-protein (Fig. 8Go). As the 3'-end of clone 42 sequence covering the entire R36 sequence is not homologous to any of these human sequences, it suggests that the 3'-end of clone 42 sequence may be species specific. An evolutionary change in this protein across species may also be implied by the relatively low similarity between mouse clone 42 and D. melanogaster CG1287.



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  		Figure 8. Model of a novel oncoprotein, the clone 42 gene product. In the DNA sequence of the PTD010 gene, an adenine nucleotide (Fig. 2AGo) is deleted at position 1034 that causes an ORF shift and stops translation at position 1086 instead of 1163 for the C25077 gene. Hence, the PTD010 gene product lacks the C-terminal portion of the sequence that remains intact in C25077 and clone 42 proteins. Potential phosphorylations sites of the clone 42 protein suggest that multiple signal-transmitting sites may occur in this protein, especially T306 and T322, as the C-terminus in PTD010 lacks these residues. Since the PTD010 gene product (319 residues) is expressed in human pituitary tumor tissue, whereas the gene product (345 residues) is found in human female fetal brain, it may suggest that the C-terminal region is important for normal biological function or that alterations at the C-terminus converts the novel function to an onco-protein. Numbers in boxes indicate amino acid residues of the clone 42 protein that are predicted as transmembrane regions.

 
In summary, clone 42 is a novel gene, in that its function has yet to be defined. It is up-regulated by GH-mediated signaling with age and is widely expressed in various tissues at a transcriptional level. Its gene product is predicted to be a transmembrane protein that may be a novel receptor. Establishing the physiological action of clone 42 is our long-term goal.


   Acknowledgments
 
We thank Dr. Leslie P. Kozak for the gift of UCP1 plasmid, Dr. Karen T. Coschigano for providing technical expertise in RNA isolation and analyses, and Dr. Linda L Bellush and Ms. Amy N. Holland for maintaining the breeding colonies of mice.


   Footnotes
 
1 This work was supported in part by the State of Ohio’s Eminent Scholar’s Program that includes a grant from Milton and Laurence Goll. Back

2 Current address: Department of Pathology, University of Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, Colorado 80262. Back

Received December 8, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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