BACKGROUND
sex hormone-binding globulin  is a multifunctional protein that influences androgen and estrogen action in humans on at least two levels.
in plasma, shbg is the major sex steroid-binding protein, regulating their availability to responsive tissues.
shbg is also an integral part of a membrane-based steroid signaling pathway in certain responsive tissues, including the prostate and breast.
the initial steps of shbg-mediated steroid signaling in the prostate and breast have been well characterized in cultured cells and tissue explants  <cit> .
unliganded shbg binds to a specific membrane receptor ; the shbg-rshbg complex is activated by subsequent binding of an appropriate androgen or estrogen, independently of the androgen or estrogen receptors  <cit> .
rshbg activation leads to a rapid increase in intracellular camp  <cit> , presumably through the action of a g protein  <cit> .
studies in cultured cells have demonstrated downstream effects that include protein kinase a activation  <cit> , increased prostate specific antigen expression  <cit> , decreased progesterone receptor expression  <cit> , induced apoptosis  <cit> , and seemingly disparate findings of reduced mcf- <dig> breast cancer cell growth  <cit>  and increased alva- <dig> prostate cancer cell growth  <cit> .
however, a biologic role of rshbg signaling in the intact prostate and breast awaits demonstration.
the source of the shbg that initializes rshbg signaling in vivo is unclear; it could be taken up from the plasma, where it is relatively abundant, or synthesized in cells in which signaling occurs.
in addition to the liver which is well known to be the source of plasma shbg, and testis , shbg protein and mrna have been demonstrated in other human tissues, including the prostate and breast  <cit> .
the relatively stable concentration of plasma shbg  <cit>  makes it a less likely source for the initiation of rshbg signaling, as the physiology of important signaling molecules depends on their variation with time.
other, more variable sources of shbg would be better candidates.
this raises the possibility that the prostate and breast themselves could control local shbg concentrations, thereby regulating rshbg activation in an autocrine/paracrine manner.
most work regarding human shbg mrna expression has been based on earlier reports of the existence of two major and two minor transcripts  <cit>  .
a downstream promoter, , regulates the expression of a major, eight-exon mrna transcript that encodes shbg .
the liver secretes shbg  <cit> ; the nascent shbg translation product is a  <dig> amino acid precursor protein which is glycosylated and cleaved at its amino terminus to remove a  <dig> amino acid long leader peptide  <cit>  leading to the secretion of mature shbg into plasma.
domains responsible for steroid binding and dimerization are encoded within exons  <dig> and  <dig> <cit> , while binding of shbg to rshbg is mediated by a decapeptide sequence encoded within exon  <dig>  <cit> .
mature shbg has an o-linked glycosylation site at thr <dig>  and n-linked sites at asn  <dig> and asn  <dig>  <cit> .
the identical eight exon transcript is also expressed in the testis, which is translated into abp  <cit> .
in addition to the shbg/abp transcript, there is a second major shbg gene transcript in the testis  derived from a second promoter , located  <dig>  kb upstream of pl  <cit> .
it possesses unique 5' end sequences, referred to here as exon 1t, and lacks exon  <dig>  transgenic mice express a human shbg species that appears to be attributable to this transcript  <cit> , however, such an isoform has not been identified in humans.
in addition to the two major transcripts, two minor human shbg gene transcripts resulting from alternative splicing of exon  <dig> have been reported  <cit> .
these include an shbg/abp-like transcript that lacks exon  <dig> , and an eight exon long transcript originating from pt that retains exon  <dig>
the lncap  and mcf- <dig>  cell lines express shbg at the mrna and protein levels and possess rshbg activity <cit> , making them attractive in vitro models for studying the effects of local shbg expression on steroid signaling and rshbg regulation.
these cell lines retain both alleles of the p <dig> gene  <cit> , located ~ <dig> kb from the distal end of the shbg gene locus on chromosome 17p <dig>  and, due to this extremely close linkage, we presumed that both shbg alleles also remain intact.
we initially intended to confirm prior reports of shbg mrna expression in these cell lines as a prelude to investigating the function of locally expressed shbg in the prostate and breast.
as positive controls for shbg mrna expression, we included the human hepg <dig>  cell line  and normal testis tissue .
in the course of that effort, we found that transcription of the human shbg gene is far more complex than had been previously reported.
we performed a detailed structural analysis of human shbg transcripts from the human lncap, mcf- <dig>  and hepg <dig> cell lines, and from normal human liver, testis, prostate, breast, and brain tissues.
rt-pcr based mapping assays revealed the existence of at least three distinct human shbg gene promoters; we determined transcriptional start sites for each.
in addition to the four previously reported transcripts, we identified and characterized  <dig> novel shbg gene transcripts resulting from alternate splicing of exons  <dig>   <dig>   <dig>  and/or  <dig>  real-time, quantitative pcr showed that shbg transcription is tissue dependent, and perhaps, cancer dependent.
we also detected a smaller sized immunoreactive shbg species in lncap, mcf- <dig> and hepg <dig> cells, which raises the possibility that at least one of these transcripts is translated.
based on our current knowledge of shbg function, these results could have important biologic implications for the modulation of rshbg activity and cellular steroid signaling.
RESULTS
shbg exon 5– <dig> rt-pcr
because the longest run of a residues within the human shbg coding region is only three, an oligo-d primer was selected for first strand cdna synthesis from lncap, mcf- <dig> and hepg <dig> cells, and from normal testis.
as a control for the initial shbg rt-pcr assays, first strand cdna was also synthesized from mcf-7-tr, a stably transfected cell line that constitutively overexpresses relatively high amounts of the contiguous exon 1l- <dig> transcript containing the human shbg/abp coding region.
a short shbg rt-pcr assay, spanning exons  <dig> through  <dig>  <cit> , was used to generate partial shbg mrna expression profiles for lncap, mcf- <dig>  hepg <dig>  testis and the mcf-7-tr control.
based upon prior reports of shbg transcription , two rt-pcr fragments were anticipated from lncap and mcf- <dig> cells, a contiguous 521-bp fragment that retains exon  <dig>  and a smaller 313-bp fragment that lacks exon  <dig>  instead, lncap and mcf- <dig> gave rise to three distinct rt-pcr fragments .
all 521-bp, 313-bp, and 176-bp rt-pcr bands were isolated, reamplified, and sequenced.
in addition to the expected 521-bp and 313-bp rt-pcr fragments, whose identities were confirmed by sequencing, we also found a 176-bp rt-pcr fragment of low abundance.
the 176-bp fragment was derived from a transcript in which exon  <dig> is directly spliced to exon  <dig> .
this same 176-bp fragment was generated from hepg <dig> and normal testis.
hepg <dig> and normal testis rt-pcr also revealed a faint, intermediate rt-pcr fragment of approximately 384-bp.
we were unable to reamplify this fragment and its identity, therefore, is not established.
as expected, the mcf-7-tr rt-pcr control gave rise only to the contiguous 521-bp fragment, undoubtedly because the highly overexpressed full-length shbg/abp cdna transcript overwhelmed the rt-pcr assay.
the relative intensities of the 521-bp and 313-bp bands varied dependent on the source of the rna.
.
shbg exon 2– <dig> rt-pcr
an exon 2– <dig> rt-pcr assay was designed to screen for alternative splicing of downstream exons, 3– <dig>  in a single reaction, intentionally bypassing the assessment of individual contributions from pl and pt.
first strand cdnas, generated from normal liver, prostate, and breast, as well as from brain .
each hepg <dig>  liver and testis rt-pcr fragment was reamplified and sequenced.
the largest fragment, 1075-bp, contained the expected contiguous exon 2– <dig> sequence.
four of the remaining eight rt-pcr fragments were comprised of sequences that were consistent with their sizes.
from largest to smallest, these transcripts lacked exon  <dig> ; exons  <dig> and  <dig> ; exons  <dig> and  <dig> ; and exons  <dig>   <dig> and  <dig> .
that exon  <dig> undergoes alternative splicing was confirmed using an rt-pcr assay that spanned exons  <dig> and  <dig> .
a prior report similarly found alternative splicing of exon  <dig>  using an rt-pcr assay that included primers specific for exons  <dig> and  <dig>  <cit> .
for reasons that are unclear, the four remaining rt-pcr fragments consistently gave sequences that were identical to those described above.
for example, we had expected the ~913-bp rt-pcr fragment to give rise to a sequence derived from a transcript lacking exon  <dig>  however, it gave the 1l- <dig> sequence lacking exon  <dig>  as described below, given our positive identification of an exon 1n- <dig> transcript lacking exon  <dig>  we suspect that technical factors  may have interfered with our ability to positively detect this specific transcript in the 2– <dig>  1l- <dig> and 1t- <dig> rt-pcr assays.
as expected, only a single, intense contiguous exon 2– <dig> rt-pcr fragment was generated from mcf-7-tr.
the same individual exon 2– <dig> rt-pcr fragments were generated from all three cell lines, albeit in unequal abundances .
identification of a novel shbg promoter and transcriptional start sites by race analysis
before assessing the splicing patterns of full-length shbg transcripts, we used a nonbiased approach to determine whether additional human shbg gene promoters exist along with pl and pt.
in the rat, an alternative upstream shbg promoter, pa, appears to share promoter elements with the rat fxr <dig> gene promoter  <cit> ; it was speculated to have an active human homologue  <cit> .
transcriptional start sites downstream of shbg gene promoters were identified using rlm-race , a modified race assay which included steps specifically designed to prime only full length transcripts that possess 5' end cap structures.
beginning with total cellular rnas isolated from lncap, hepg <dig>  and normal testis, multiple rlm-race bands were generated from each sample , and each band was reamplified and sequenced.
the largest hepg <dig> band was actually a mixture of two closely sized race fragments.
distinct exon 1l 5' end boundaries mapped to a residues respectively located 79-  and 38-nt  upstream of the atg start codon, indicating that pl utilizes at least two transcriptional initiation sites .
the proximal initiation site is consistent with positions of the major exon 1l rat and rabbit shbg transcriptional initiation sites  <cit> .
our results differ from those human exon 1l transcriptional start sites originally determined  <cit> , and with those mapped in the liver and kidney of mice expressing a human shbg transgene  <cit> .
the largest rlm-race band from normal testis was also a mixture of two similarly sized fragments, both of which contained exon 1t sequences .
the longer sequence extended to a g residue located 183-nt upstream of the exon 1t splice donor site , whereas the shorter fragment extended to a t residue located 161-nt upstream  of the splice donor site.
these results are consistent with primer extension studies in the testis of a mouse expressing a human shbg transgene, where major exon 1t initiation sites were located  <dig>   <dig>  and 186-nt upstream of the exon 1t splice donor site  <cit> .
our results confirm that exon 1t lacks a potential atg start codon.
lncap gave rise to a race fragment consisting of a novel, 164-bp sequence  spliced directly to shbg exon  <dig>  this sequence maps to an uninterrupted genomic locus located  <dig> kb upstream of pl on chromosome 17p <dig> , within the first intron of the fragile x mental retardation-related  <dig>  gene, and is referred to here as shbg exon 1n .
exon 1n is distinct from the human counterpart of rat exon 1a  , located on the opposite side of the fxr <dig> gene promoter.
we confirmed the 1n transcript initiation site in lncap cells in a second race assay, using a downstream primer complementary to 3' end sequences within exon 1n and the same 5' adaptor based primer .
a short exon 1n- <dig> rt-pcr assay was also performed on lncap, mcf- <dig>  and hepg <dig> to confirm that exon 1n is incorporated into mature shbg mrna transcripts.
a single rt-pcr fragment of the expected size was generated from lncap and mcf- <dig>  but not from hepg <dig> .
all other race bands generated from lncap, hepg <dig> and normal testis were either comprised of shbg sequences that had recombined with sequences from other chromosomes or were not specific for shbg, and were considered to be experimental artifacts.
this experimental approach does not rule out the possibility that additional human shbg gene promoters may still exist.
shbg exon 1l-, 1t-, and 1n- <dig> rt-pcr
a comprehensive structural analysis of human shbg gene transcripts was performed using exon 1l- <dig>  1t- <dig>  and 1n- <dig> rt-pcr assays.
these incorporated upstream primers specific for either exons 1l, 1t, or 1n and a common, downstream exon  <dig> primer.
for these rt-pcr experiments, because exon  <dig> is the last described exon in the human shbg gene, and it contains appropriate transcriptional processing sequences, the use of a reverse primer complementary to exon  <dig> sequences demonstrates the likely presence of full length transcripts.
for the most part, counterparts of all exon 2– <dig> assay rt-pcr fragments appeared in one or more of the exon 1– <dig> assays .
overall, normal liver, prostate, and breast expressed a lower proportion of alternatively spliced shbg transcripts than did the hepg <dig>  lncap, and mcf- <dig> cell lines.
nine individual rt-pcr fragments were produced in the exon 1l- <dig> assay .
those generated from hepg <dig> were sequenced, as were all the  <dig> bp fragments.
the sizes of six of the nine hepg <dig> fragments were consistent with their sequences, whereas three were not, and remain unattributable.
sequence analysis of all 1130-bp fragments confirmed that they were derived from the contiguous shbg/abp transcript.
this fragment was the sole detectable 1l- <dig> species in prostate, breast and brain.
hepg <dig> also displayed 1l- <dig> rt-pcr fragments corresponding to smaller sized, alternatively spliced transcripts  that lacked exon  <dig>  exons  <dig> and  <dig> , exons  <dig> and  <dig> , exons  <dig>   <dig>  and  <dig> , and a novel, 463-bp rt-pcr fragment.
the 463-bp fragment was comprised of exon 1l,  <dig>   <dig>  and  <dig> sequences , representing a transcript lacking exons 4– <dig>  to our knowledge, this is the first account of a human shbg transcript that lacks exon  <dig> sequences.
alternatively spliced 1l- <dig> species were also observed in liver, testis, mcf- <dig> and lncap .
the sequences obtained for the remaining three different sized rt-pcr fragments unexpectedly gave sequences that were identical to specific shbg transcripts described above.
because of the incongruence of their sizes and sequences, and despite repeated attempts at their resolution, their significance remains uncertain.
the exon 1t- <dig> assay produced a total of ten individual rt-pcr fragments .
those generated from lncap and testis were reamplified and sequenced, as were all of the  <dig> bp fragments.
six of the ten fragments lncap and testis fragments gave sequences that corresponded with their sizes.
as expected, the 943-bp fragment, corresponding to the major exon 1t- <dig> transcript lacking exon  <dig>  was the most abundant testis transcript.
this transcript was found in all samples other than for prostate and breast.
the contiguous exon 1t- <dig> transcript  was present in all samples tested.
alternatively spliced exon 1t- <dig> transcripts lacking exons  <dig> and  <dig> ,  <dig> and  <dig> , and  <dig>   <dig>  and  <dig>  were also detected.
testis and liver generated a very weak 484-bp 1t- <dig> fragment that was found by sequence analysis to consist only of exons 1t,  <dig>   <dig>  and  <dig> .
of the four remaining 1t- <dig> rt-pcr fragments, the smallest was unrelated to any shbg transcript, while the remaining three fragments gave previously characterized shbg transcript sequences that were incongruent with their sizes.
sequence analysis confirmed that all  <dig> bp fragments from the remaining samples corresponded to the contiguous 1t- <dig> transcript.
furthermore, the sequences of selected abundant bands from the 1t- <dig> assay , also confirmed their structures.
the exon 1n- <dig> assay produced a total of nine individual rt-pcr fragments .
rt-pcr fragments from lncap and mcf- <dig> were reamplified and sequenced.
six of the nine fragments gave sequences that corresponded with their sizes.
the contiguous 1146-bp 1n- <dig> fragment  was present in lncap and mcf- <dig>  and was faint in testis and prostate.
the third largest fragment was found to consist of a mixed sequence derived from two transcripts, an appropriately sized 984-bp fragment that had undergone splicing of exon  <dig> alone  and an aberrantly migrating fragment that lacks only exon  <dig>  this result suggests that transcripts lacking exon  <dig> may have gone undetected in the 1l- <dig> and 1t- <dig> assays if their sequence signals were drowned out by more abundant co-migrating species.
the 1n- <dig> assay also generated appropriately sized fragments corresponding to transcripts that have undergone splicing of exon  <dig> , and exons  <dig> and  <dig> ,  <dig> and  <dig> ,  <dig>   <dig>  and  <dig> , and exons  <dig>   <dig>   <dig>  and  <dig>  .
the reproducibility of the testis and hepg <dig> assays was inconsistent, likely due to the low abundance of 1n- <dig> transcripts in these samples.
quantitative pcr  analysis of shbg transcript abundance in human tissues and cell lines
four independent real time, qpcr assays were performed to determine total relative shbg mrna abundance, and the relative abundance of transcripts derived from each of the three promoters.
total shbg transcript abundance was measured by targeting common sequences within exons  <dig> and  <dig> that are present within all the shbg transcripts identified in this study.
contributions from individual promoters were measured using qpcr assays specific for exons 1l, 1t, and 1n, respectively.
the samples were normalized against gapdh expression.
overall, the exon 1l, and 1t, qpcr assays gave results that were comparable to the three individual exon 1– <dig> rt-pcr assays.
among the normal tissues, overall shbg gene transcription, as measured by the exon 2– <dig> qpcr assay, was highest in liver and testis.
transcripts containing exon 1l were most abundant in liver, whereas those containing exon 1t were most abundant in testis.
all normal tissues, other than liver, expressed transcripts containing exon 1n.
compared to their respective parental tissues, the three cancer cell lines all displayed reduced expression of exon 1l-containing transcripts, whereas there were varied differences in the abundance of exon 1t- and 1n-containing transcripts.
apparent discrepancies between certain exon 1n qpcr and rt-pcr results could signify that novel 3' end sequences may be incorporated into a fraction of shbg gene transcripts; this would have gone undetected here due to our exclusive use of an exon 8-based downstream primer in longer range rt-pcr assays.
thus, actual human shbg gene expression may be even more complex than is presented here.
initial western blot analysis of shbg protein expression in hepg <dig>  lncap, and mcf- <dig> cells
we addressed the question of the existence of shbg isoforms in hepg <dig>  lncap, and mcf- <dig> cells in initial western blot studies.
immunoblot analysis of solubilized cellular proteins separated by sds-page, was performed using a polyclonal rabbit anti-human shbg antibody .
western blot analysis  shows that all three cell lines express an immunoreactive protein of the same molecular weight as purified shbg.
a lower molecular weight species of 33– <dig> kd was also detected .
to our knowledge, this is the first demonstration that such a lower molecular weight shbg species exists in these cell lines.
since immunoreactive shbg was not detected in the fetal bovine serum used in the prepared media , we believe that this lower molecular weight species is either an isoform derived from one of the alternatively spliced shbg transcripts, or a proteolytic fragment of shbg itself.
assigning one of the transcripts described above to this species is difficult without further study, since shbg has multiple glycoslyation sites that could increase its observed size by up to  <dig> kilodaltons, and a  <dig> amino acid long signal peptide at its amino terminus that may or may not be retained.
potential candidates include the 1l transcript lacking exons  <dig> and  <dig> which encodes an unmodified protein of  <dig>  kilodaltons.
if it exists, the exon 1l transcript lacking exon  <dig> encodes an unmodified protein of  <dig>  kilodaltons; without the signal peptide it would have a molecular weight of  <dig>  kilodaltons.
further studies should identify the origin of this novel smaller species.
compared to hepg <dig> and lncap, the appearance of multiple larger species in mcf- <dig> is likely due to differential glycosylation of shbg itself  <cit> .
discussion
it is evident from these results that the current view of human shbg gene structure, transcription, and, perhaps, protein expression requires modification.
in addition to the two previously described human shbg gene promoters, pl, and pt, we provide evidence for a novel, third upstream promoter, pn.
combined, these three promoters are responsible for generating at least  <dig> unique transcripts .
to our knowledge,  <dig> of these  <dig> shbg gene transcripts are novel, with the smaller sized shbg mrnas resulting from alternative splicing patterns that target exons  <dig>   <dig>   <dig>  and/or  <dig>
alternative splicing of human shbg transcripts
certain nonannotated data in earlier studies on human shbg gene expression are consistent with the existence of some of the novel, alternatively spliced human shbg transcripts in this communication.
using an identical exon 5– <dig> rt-pcr assay, an uncharacterized small rt-pcr fragment was generated from zr-75- <dig> and mda-mb- <dig> cells , whose molecular size is consistent with the 176-bp fragment lacking exons  <dig> and  <dig> in our exon 5– <dig> rt-pcr assay .
this fragment is also present in our mcf- <dig> assay but was not apparent in theirs.
forges et al.
showed that human testis expresses the four earlier described shbg transcripts; their data also include minor rt-pcr fragments that correspond to novel transcripts characterized in the present study.
selva et al.
appear to generate at least one faint intermediate band between the contiguous exon 1t- <dig> and the exon  <dig> lacking rt-pcr fragments in human testis and sperm in their exon 1t- <dig> assay .
more recently, ng et al.
used an exon 3– <dig> rt-pcr assay to demonstrate splicing of exon  <dig> alone  <cit> .
in view of this new profile of human shbg transcription and alternative splicing, conclusions from prior studies on shbg expression based upon short-range rt-pcr assays may require reevaluation.
expression of the shbg/abp transcript
the notion that shbg protein itself is expressed in certain hormonally responsive tissues is supported by the transcriptional profiles presented in this study, and our western blot results.
human prostate  <cit>  and breast  <cit> , and specific regions of the brain  <cit>  have previously been shown to display immunoreactive shbg protein.
our 1l- <dig> rt-pcr assay showed that each of these tissues expresses the shbg/abp transcript.
compared to liver, the relative low expression of the transcript encoding shbg in breast and prostate is consistent with a local effect/need for shbg in acting on rshbg.
extending this speculation, fibulin-1d and fibulin- <dig> have been shown to sequester plasma shbg in the uterine stromal cells of transgenic mice, with a preference for steroid-bound shbg  <cit> .
such an interaction could be envisioned as regulating local rshbg signaling and/or sex steroid delivery in the human prostate, where shbg expression predominates in luminal epithelial cells and rshbg activity is highest in stromal cells  <cit> .
it has been recently reported that shbg acts to actively transport steroids into cells by binding of the endocytic receptor, megalin  <cit> , though this finding has been disputed  <cit> .
should megalin serve such a function in the breast and prostate, then locally expressed shbg could be a major player in steroid uptake in these tissues.
intracellular shbg might also bind specific sex steroids, thereby influencing ar or er activation.
therefore, locally expressed shbg could play a role in a number of different biologic functions related to sex steroids.
biologic significance of alternatively spliced shbg gene transcripts
we suspect that only specific transcripts derived from pl are likely candidates to encode stably translated shbg isoforms.
the exon  <dig> reading frame is retained in pl derived transcripts that lack exon  <dig> alone, exons  <dig> and  <dig>  and exons  <dig>   <dig>  and  <dig>  the remaining pl derived transcripts, which present frameshifts within exon  <dig>  appear less likely to be stably translated; an e <dig> snp variant of shbg, possessing a premature termination codon, is rapidly degraded  <cit>  suggesting that motifs encoded within exon  <dig> confer protein stability  <cit> .
we do not rule out the possibility that the exon 1l- <dig> transcript lacking exon  <dig> exists and went undetected due to aberrantly comigrating sequences, considering that we detected an exon 1n- <dig> transcript that lacks exon  <dig> from a mixture of comigrating rt-pcr fragments.
supporting this contention is a prior report that showed hepg <dig> transcripts to undergo splicing of exon  <dig> alone  <cit> .
based on its size alone, the 1l- <dig> transcript lacking exon  <dig> could encode the smaller, immunoreactive species recently described in human sperm  <cit> .
based upon their sequences, conventional translational initiation events can not explain how human pn- or pt- derived transcripts are translated into stable shbg isoforms.
the first potential aug start codon within exon 1n heads a short, four amino acid open reading frame.
the subsequent aug codon lies at the beginning of a  <dig> amino acid open reading frame; a termination codon precludes its merge into the exon  <dig> shbg/abp reading frame.
exon 1t is devoid of an aug start codon, the first potential aug start codon within exon  <dig> of pt- derived transcripts occurs at the beginning of a short open reading frame consisting of only  <dig> amino acids.
for such transcripts, it has been suggested that translation may initiate within exon  <dig> at a downstream aug codon that shares the shbg/abp open reading frame, or that they may utilize alternative, rare start codons within exon 1t  <cit> .
we note that, beginning with the cug at position + <dig>  there are eight possible alternative cug, uug, gug, or aua start codons  <cit>  within the long exon 1t open reading frame that merges with the shbg/abp reading frame in exon  <dig>  more work is required to determine the biologic fates of transcripts originating from pt and pn.
functionally, as has been seen with other genes  <cit> , it is feasible that nontranslated transcripts originating from pt and pn could be regulatory in nature, affecting the expression of those that do encode shbg and stable shbg isoforms.
the 1l- <dig> transcripts lacking exons  <dig> and  <dig> or exon  <dig> alone could encode the smaller shbg species we detected in hepg <dig>  lncap and mcf- <dig> cells.
a similar sized species was not identified in affinity purified shbg from human plasma, hence, it might not bind steroids, and/or it may remain intracellular.
the existence of an intracellular shbg species that does not bind steroid, but retains the rshbg membrane-binding motif within exon  <dig>  <cit>  would be quite exciting, as it could potentially affect rshbg signaling, as well as other shbg mediated properties.
in addition to our finding of a smaller shbg species in cell lines, human hypothalamus and cerebrospinal fluid have been shown to contain an immunoreactive shbg species which is  <dig> -kd smaller than that of serum shbg  <cit> .
lower molecular weight species have been isolated from human male genital tissue and myocardium  <cit> .
taken together, these results suggest that a deeper investigation of alternatively spliced transcripts originating from pl may explain these findings.
human shbg gene structure
the existence of human shbg gene transcripts originating from pt and pn fits a larger picture in which the mammalian shbg gene is in a state of evolutionary flux, 'hijacking' promoter elements from adjacent upstream genes on chromosome 17p <dig>  that are transcribed in the opposite orientation .
pt, which is located  <dig>  kb upstream of pl, shares regulatory elements with the adjacent polyamine n-acetyltransferase  gene.
based on chromosomal mapping of shbg exon 1n, pn is located within intron  <dig> of the fxr <dig> gene.
such overlapping transcriptional units and bi-directional promoter arrangements have been described for a number of genes; computational studies of gene organization predict them to occur frequently within the human genome  <cit> .
the rat possesses homologous exon 1n sequences , but these have not been reported in mature rat shbg transcripts.
while prior work suggested that certain orthologous sequences might be incorporated into mature human transcripts, using race assays, we did not specifically detect the existence of a human homologue of rat pa  <cit> .
furthermore, the putative human exon 1a region contains no potential start codons , thus, transcripts originating from a human pa would run into the same functional and translational questions as do transcripts derived from pt.
relative abundance of human shbg transcripts in tissues and cell lines
transcripts derived from pl, pt, and pn are expressed differentially in normal prostate, breast, liver, and testis.
the relative expression of the shbg/abp transcript is consistent with our hypothesis regarding local generation of shbg vs. the utilization of plasma shbg- that immediate, rapid effects of shbg, including those mediated by the rshbg signaling pathway, are likely to be regulated through local expression of shbg within responsive tissues; the concentration and/or availability of plasma shbg is not responsive to shbg needs in particular sites.
each of the cell lines we examined had a reduced abundance of total 1l transcripts as compared to their corresponding normal tissues, and more specifically this was the case for the shbg/abp transcript.
furthermore, the cancer cell lines examined in these studies had a greater overall abundance of alternatively spliced pl derived transcripts than did their corresponding normal tissues.
if shbg gene expression is indeed modulated in prostate and breast cancer, this could alter their androgen and estrogen responsiveness.
CONCLUSIONS
in conclusion, our findings provide a broad and detailed picture of human shbg transcript structure and expression.
at the same time, they also raise many new and important questions, including the viability of novel alternatively spliced transcripts, their functions in hormonally responsive tissues, including the breast and prostate, and the role that aberrant shbg gene expression may play in cancer.

 sex hormone-binding globulin is a multifunctional protein that influences androgen and estrogen action in humans on at least two levels. in plasma, shbg is the major sex steroid-binding protein, regulating their availability to responsive tissues. shbg is also an integral part of a membrane-based steroid signaling pathway in certain responsive tissues, including the prostate and breast. the initial steps of shbg-mediated steroid signaling in the prostate and breast have been well characterized in cultured cells and tissue explants. unliganded shbg binds to a specific membrane receptor; the shbg-rshbg complex is activated by subsequent binding of an appropriate androgen or estrogen, independently of the androgen or estrogen receptors. rshbg activation leads to a rapid increase in intracellular camp, presumably through the action of a g protein. studies in cultured cells have demonstrated downstream effects that include protein kinase a activation, increased prostate specific antigen expression, decreased progesterone receptor expression, induced apoptosis, and seemingly disparate findings of reduced mcf- breast cancer cell growth and increased alva- prostate cancer cell growth. however, a biologic role of rshbg signaling in the intact prostate and breast awaits demonstration. the source of the shbg that initializes rshbg signaling in vivo is unclear; it could be taken up from the plasma, where it is relatively abundant, or synthesized in cells in which signaling occurs. in addition to the liver which is well known to be the source of plasma shbg, and testis, shbg protein and mrna have been demonstrated in other human tissues, including the prostate and breast. the relatively stable concentration of plasma shbg makes it a less likely source for the initiation of rshbg signaling, as the physiology of important signaling molecules depends on their variation with time. other, more variable sources of shbg would be better candidates. this raises the possibility that the prostate and breast themselves could control local shbg concentrations, thereby regulating rshbg activation in an autocrine/paracrine manner. most work regarding human shbg mrna expression has been based on earlier reports of the existence of two major and two minor transcripts. a downstream promoter,, regulates the expression of a major, eight-exon mrna transcript that encodes shbg. the liver secretes shbg; the nascent shbg translation product is a amino acid precursor protein which is glycosylated and cleaved at its amino terminus to remove a amino acid long leader peptide leading to the secretion of mature shbg into plasma. domains responsible for steroid binding and dimerization are encoded within exons and, while binding of shbg to rshbg is mediated by a decapeptide sequence encoded within exon. mature shbg has an o-linked glycosylation site at thr and n-linked sites at asn and asn. the identical eight exon transcript is also expressed in the testis, which is translated into abp. in addition to the shbg/abp transcript, there is a second major shbg gene transcript in the testis derived from a second promoter, located kb upstream of pl. it possesses unique 5' end sequences, referred to here as exon 1t, and lacks exon transgenic mice express a human shbg species that appears to be attributable to this transcript, however, such an isoform has not been identified in humans. in addition to the two major transcripts, two minor human shbg gene transcripts resulting from alternative splicing of exon have been reported. these include an shbg/abp-like transcript that lacks exon, and an eight exon long transcript originating from pt that retains exon the lncap and mcf- cell lines express shbg at the mrna and protein levels and possess rshbg activity, making them attractive in vitro models for studying the effects of local shbg expression on steroid signaling and rshbg regulation. these cell lines retain both alleles of the p gene, located ~ kb from the distal end of the shbg gene locus on chromosome 17p and, due to this extremely close linkage, we presumed that both shbg alleles also remain intact. we initially intended to confirm prior reports of shbg mrna expression in these cell lines as a prelude to investigating the function of locally expressed shbg in the prostate and breast. as positive controls for shbg mrna expression, we included the human hepg cell line and normal testis tissue. in the course of that effort, we found that transcription of the human shbg gene is far more complex than had been previously reported. we performed a detailed structural analysis of human shbg transcripts from the human lncap, mcf- and hepg cell lines, and from normal human liver, testis, prostate, breast, and brain tissues. rt-pcr based mapping assays revealed the existence of at least three distinct human shbg gene promoters; we determined transcriptional start sites for each. in addition to the four previously reported transcripts, we identified and characterized novel shbg gene transcripts resulting from alternate splicing of exons and/or real-time, quantitative pcr showed that shbg transcription is tissue dependent, and perhaps, cancer dependent. we also detected a smaller sized immunoreactive shbg species in lncap, mcf- and hepg cells, which raises the possibility that at least one of these transcripts is translated. based on our current knowledge of shbg function, these results could have important biologic implications for the modulation of rshbg activity and cellular steroid signaling.  shbg exon 5– rt-pcr because the longest run of a residues within the human shbg coding region is only three, an oligo-d primer was selected for first strand cdna synthesis from lncap, mcf- and hepg cells, and from normal testis. as a control for the initial shbg rt-pcr assays, first strand cdna was also synthesized from mcf-7-tr, a stably transfected cell line that constitutively overexpresses relatively high amounts of the contiguous exon 1l- transcript containing the human shbg/abp coding region. a short shbg rt-pcr assay, spanning exons through, was used to generate partial shbg mrna expression profiles for lncap, mcf- hepg testis and the mcf-7-tr control. based upon prior reports of shbg transcription, two rt-pcr fragments were anticipated from lncap and mcf- cells, a contiguous 521-bp fragment that retains exon and a smaller 313-bp fragment that lacks exon instead, lncap and mcf- gave rise to three distinct rt-pcr fragments. all 521-bp, 313-bp, and 176-bp rt-pcr bands were isolated, reamplified, and sequenced. in addition to the expected 521-bp and 313-bp rt-pcr fragments, whose identities were confirmed by sequencing, we also found a 176-bp rt-pcr fragment of low abundance. the 176-bp fragment was derived from a transcript in which exon is directly spliced to exon. this same 176-bp fragment was generated from hepg and normal testis. hepg and normal testis rt-pcr also revealed a faint, intermediate rt-pcr fragment of approximately 384-bp. we were unable to reamplify this fragment and its identity, therefore, is not established. as expected, the mcf-7-tr rt-pcr control gave rise only to the contiguous 521-bp fragment, undoubtedly because the highly overexpressed full-length shbg/abp cdna transcript overwhelmed the rt-pcr assay. the relative intensities of the 521-bp and 313-bp bands varied dependent on the source of the rna.. shbg exon 2– rt-pcr an exon 2– rt-pcr assay was designed to screen for alternative splicing of downstream exons, 3– in a single reaction, intentionally bypassing the assessment of individual contributions from pl and pt. first strand cdnas, generated from normal liver, prostate, and breast, as well as from brain. each hepg liver and testis rt-pcr fragment was reamplified and sequenced. the largest fragment, 1075-bp, contained the expected contiguous exon 2– sequence. four of the remaining eight rt-pcr fragments were comprised of sequences that were consistent with their sizes. from largest to smallest, these transcripts lacked exon; exons and; exons and; and exons and. that exon undergoes alternative splicing was confirmed using an rt-pcr assay that spanned exons and. a prior report similarly found alternative splicing of exon using an rt-pcr assay that included primers specific for exons and. for reasons that are unclear, the four remaining rt-pcr fragments consistently gave sequences that were identical to those described above. for example, we had expected the ~913-bp rt-pcr fragment to give rise to a sequence derived from a transcript lacking exon however, it gave the 1l- sequence lacking exon as described below, given our positive identification of an exon 1n- transcript lacking exon we suspect that technical factors may have interfered with our ability to positively detect this specific transcript in the 2– 1l- and 1t- rt-pcr assays. as expected, only a single, intense contiguous exon 2– rt-pcr fragment was generated from mcf-7-tr. the same individual exon 2– rt-pcr fragments were generated from all three cell lines, albeit in unequal abundances. identification of a novel shbg promoter and transcriptional start sites by race analysis before assessing the splicing patterns of full-length shbg transcripts, we used a nonbiased approach to determine whether additional human shbg gene promoters exist along with pl and pt. in the rat, an alternative upstream shbg promoter, pa, appears to share promoter elements with the rat fxr gene promoter; it was speculated to have an active human homologue. transcriptional start sites downstream of shbg gene promoters were identified using rlm-race, a modified race assay which included steps specifically designed to prime only full length transcripts that possess 5' end cap structures. beginning with total cellular rnas isolated from lncap, hepg and normal testis, multiple rlm-race bands were generated from each sample, and each band was reamplified and sequenced. the largest hepg band was actually a mixture of two closely sized race fragments. distinct exon 1l 5' end boundaries mapped to a residues respectively located 79- and 38-nt upstream of the atg start codon, indicating that pl utilizes at least two transcriptional initiation sites. the proximal initiation site is consistent with positions of the major exon 1l rat and rabbit shbg transcriptional initiation sites. our results differ from those human exon 1l transcriptional start sites originally determined, and with those mapped in the liver and kidney of mice expressing a human shbg transgene. the largest rlm-race band from normal testis was also a mixture of two similarly sized fragments, both of which contained exon 1t sequences. the longer sequence extended to a g residue located 183-nt upstream of the exon 1t splice donor site, whereas the shorter fragment extended to a t residue located 161-nt upstream of the splice donor site. these results are consistent with primer extension studies in the testis of a mouse expressing a human shbg transgene, where major exon 1t initiation sites were located and 186-nt upstream of the exon 1t splice donor site. our results confirm that exon 1t lacks a potential atg start codon. lncap gave rise to a race fragment consisting of a novel, 164-bp sequence spliced directly to shbg exon this sequence maps to an uninterrupted genomic locus located kb upstream of pl on chromosome 17p, within the first intron of the fragile x mental retardation-related gene, and is referred to here as shbg exon 1n. exon 1n is distinct from the human counterpart of rat exon 1a, located on the opposite side of the fxr gene promoter. we confirmed the 1n transcript initiation site in lncap cells in a second race assay, using a downstream primer complementary to 3' end sequences within exon 1n and the same 5' adaptor based primer. a short exon 1n- rt-pcr assay was also performed on lncap, mcf- and hepg to confirm that exon 1n is incorporated into mature shbg mrna transcripts. a single rt-pcr fragment of the expected size was generated from lncap and mcf- but not from hepg. all other race bands generated from lncap, hepg and normal testis were either comprised of shbg sequences that had recombined with sequences from other chromosomes or were not specific for shbg, and were considered to be experimental artifacts. this experimental approach does not rule out the possibility that additional human shbg gene promoters may still exist. shbg exon 1l-, 1t-, and 1n- rt-pcr a comprehensive structural analysis of human shbg gene transcripts was performed using exon 1l- 1t- and 1n- rt-pcr assays. these incorporated upstream primers specific for either exons 1l, 1t, or 1n and a common, downstream exon primer. for these rt-pcr experiments, because exon is the last described exon in the human shbg gene, and it contains appropriate transcriptional processing sequences, the use of a reverse primer complementary to exon sequences demonstrates the likely presence of full length transcripts. for the most part, counterparts of all exon 2– assay rt-pcr fragments appeared in one or more of the exon 1– assays. overall, normal liver, prostate, and breast expressed a lower proportion of alternatively spliced shbg transcripts than did the hepg lncap, and mcf- cell lines. nine individual rt-pcr fragments were produced in the exon 1l- assay. those generated from hepg were sequenced, as were all the bp fragments. the sizes of six of the nine hepg fragments were consistent with their sequences, whereas three were not, and remain unattributable. sequence analysis of all 1130-bp fragments confirmed that they were derived from the contiguous shbg/abp transcript. this fragment was the sole detectable 1l- species in prostate, breast and brain. hepg also displayed 1l- rt-pcr fragments corresponding to smaller sized, alternatively spliced transcripts that lacked exon exons and, exons and, exons and, and a novel, 463-bp rt-pcr fragment. the 463-bp fragment was comprised of exon 1l, and sequences, representing a transcript lacking exons 4– to our knowledge, this is the first account of a human shbg transcript that lacks exon sequences. alternatively spliced 1l- species were also observed in liver, testis, mcf- and lncap. the sequences obtained for the remaining three different sized rt-pcr fragments unexpectedly gave sequences that were identical to specific shbg transcripts described above. because of the incongruence of their sizes and sequences, and despite repeated attempts at their resolution, their significance remains uncertain. the exon 1t- assay produced a total of ten individual rt-pcr fragments. those generated from lncap and testis were reamplified and sequenced, as were all of the bp fragments. six of the ten fragments lncap and testis fragments gave sequences that corresponded with their sizes. as expected, the 943-bp fragment, corresponding to the major exon 1t- transcript lacking exon was the most abundant testis transcript. this transcript was found in all samples other than for prostate and breast. the contiguous exon 1t- transcript was present in all samples tested. alternatively spliced exon 1t- transcripts lacking exons and, and, and and were also detected. testis and liver generated a very weak 484-bp 1t- fragment that was found by sequence analysis to consist only of exons 1t, and. of the four remaining 1t- rt-pcr fragments, the smallest was unrelated to any shbg transcript, while the remaining three fragments gave previously characterized shbg transcript sequences that were incongruent with their sizes. sequence analysis confirmed that all bp fragments from the remaining samples corresponded to the contiguous 1t- transcript. furthermore, the sequences of selected abundant bands from the 1t- assay, also confirmed their structures. the exon 1n- assay produced a total of nine individual rt-pcr fragments. rt-pcr fragments from lncap and mcf- were reamplified and sequenced. six of the nine fragments gave sequences that corresponded with their sizes. the contiguous 1146-bp 1n- fragment was present in lncap and mcf- and was faint in testis and prostate. the third largest fragment was found to consist of a mixed sequence derived from two transcripts, an appropriately sized 984-bp fragment that had undergone splicing of exon alone and an aberrantly migrating fragment that lacks only exon this result suggests that transcripts lacking exon may have gone undetected in the 1l- and 1t- assays if their sequence signals were drowned out by more abundant co-migrating species. the 1n- assay also generated appropriately sized fragments corresponding to transcripts that have undergone splicing of exon, and exons and, and, and, and exons and. the reproducibility of the testis and hepg assays was inconsistent, likely due to the low abundance of 1n- transcripts in these samples. quantitative pcr analysis of shbg transcript abundance in human tissues and cell lines four independent real time, qpcr assays were performed to determine total relative shbg mrna abundance, and the relative abundance of transcripts derived from each of the three promoters. total shbg transcript abundance was measured by targeting common sequences within exons and that are present within all the shbg transcripts identified in this study. contributions from individual promoters were measured using qpcr assays specific for exons 1l, 1t, and 1n, respectively. the samples were normalized against gapdh expression. overall, the exon 1l, and 1t, qpcr assays gave results that were comparable to the three individual exon 1– rt-pcr assays. among the normal tissues, overall shbg gene transcription, as measured by the exon 2– qpcr assay, was highest in liver and testis. transcripts containing exon 1l were most abundant in liver, whereas those containing exon 1t were most abundant in testis. all normal tissues, other than liver, expressed transcripts containing exon 1n. compared to their respective parental tissues, the three cancer cell lines all displayed reduced expression of exon 1l-containing transcripts, whereas there were varied differences in the abundance of exon 1t- and 1n-containing transcripts. apparent discrepancies between certain exon 1n qpcr and rt-pcr results could signify that novel 3' end sequences may be incorporated into a fraction of shbg gene transcripts; this would have gone undetected here due to our exclusive use of an exon 8-based downstream primer in longer range rt-pcr assays. thus, actual human shbg gene expression may be even more complex than is presented here. initial western blot analysis of shbg protein expression in hepg lncap, and mcf- cells we addressed the question of the existence of shbg isoforms in hepg lncap, and mcf- cells in initial western blot studies. immunoblot analysis of solubilized cellular proteins separated by sds-page, was performed using a polyclonal rabbit anti-human shbg antibody. western blot analysis shows that all three cell lines express an immunoreactive protein of the same molecular weight as purified shbg. a lower molecular weight species of 33– kd was also detected. to our knowledge, this is the first demonstration that such a lower molecular weight shbg species exists in these cell lines. since immunoreactive shbg was not detected in the fetal bovine serum used in the prepared media, we believe that this lower molecular weight species is either an isoform derived from one of the alternatively spliced shbg transcripts, or a proteolytic fragment of shbg itself. assigning one of the transcripts described above to this species is difficult without further study, since shbg has multiple glycoslyation sites that could increase its observed size by up to kilodaltons, and a amino acid long signal peptide at its amino terminus that may or may not be retained. potential candidates include the 1l transcript lacking exons and which encodes an unmodified protein of kilodaltons. if it exists, the exon 1l transcript lacking exon encodes an unmodified protein of kilodaltons; without the signal peptide it would have a molecular weight of kilodaltons. further studies should identify the origin of this novel smaller species. compared to hepg and lncap, the appearance of multiple larger species in mcf- is likely due to differential glycosylation of shbg itself. discussion it is evident from these results that the current view of human shbg gene structure, transcription, and, perhaps, protein expression requires modification. in addition to the two previously described human shbg gene promoters, pl, and pt, we provide evidence for a novel, third upstream promoter, pn. combined, these three promoters are responsible for generating at least unique transcripts. to our knowledge, of these shbg gene transcripts are novel, with the smaller sized shbg mrnas resulting from alternative splicing patterns that target exons and/or alternative splicing of human shbg transcripts certain nonannotated data in earlier studies on human shbg gene expression are consistent with the existence of some of the novel, alternatively spliced human shbg transcripts in this communication. using an identical exon 5– rt-pcr assay, an uncharacterized small rt-pcr fragment was generated from zr-75- and mda-mb- cells, whose molecular size is consistent with the 176-bp fragment lacking exons and in our exon 5– rt-pcr assay. this fragment is also present in our mcf- assay but was not apparent in theirs. forges et al. showed that human testis expresses the four earlier described shbg transcripts; their data also include minor rt-pcr fragments that correspond to novel transcripts characterized in the present study. selva et al. appear to generate at least one faint intermediate band between the contiguous exon 1t- and the exon lacking rt-pcr fragments in human testis and sperm in their exon 1t- assay. more recently, ng et al. used an exon 3– rt-pcr assay to demonstrate splicing of exon alone. in view of this new profile of human shbg transcription and alternative splicing, conclusions from prior studies on shbg expression based upon short-range rt-pcr assays may require reevaluation. expression of the shbg/abp transcript the notion that shbg protein itself is expressed in certain hormonally responsive tissues is supported by the transcriptional profiles presented in this study, and our western blot results. human prostate and breast, and specific regions of the brain have previously been shown to display immunoreactive shbg protein. our 1l- rt-pcr assay showed that each of these tissues expresses the shbg/abp transcript. compared to liver, the relative low expression of the transcript encoding shbg in breast and prostate is consistent with a local effect/need for shbg in acting on rshbg. extending this speculation, fibulin-1d and fibulin- have been shown to sequester plasma shbg in the uterine stromal cells of transgenic mice, with a preference for steroid-bound shbg. such an interaction could be envisioned as regulating local rshbg signaling and/or sex steroid delivery in the human prostate, where shbg expression predominates in luminal epithelial cells and rshbg activity is highest in stromal cells. it has been recently reported that shbg acts to actively transport steroids into cells by binding of the endocytic receptor, megalin, though this finding has been disputed. should megalin serve such a function in the breast and prostate, then locally expressed shbg could be a major player in steroid uptake in these tissues. intracellular shbg might also bind specific sex steroids, thereby influencing ar or er activation. therefore, locally expressed shbg could play a role in a number of different biologic functions related to sex steroids. biologic significance of alternatively spliced shbg gene transcripts we suspect that only specific transcripts derived from pl are likely candidates to encode stably translated shbg isoforms. the exon reading frame is retained in pl derived transcripts that lack exon alone, exons and and exons and the remaining pl derived transcripts, which present frameshifts within exon appear less likely to be stably translated; an e snp variant of shbg, possessing a premature termination codon, is rapidly degraded suggesting that motifs encoded within exon confer protein stability. we do not rule out the possibility that the exon 1l- transcript lacking exon exists and went undetected due to aberrantly comigrating sequences, considering that we detected an exon 1n- transcript that lacks exon from a mixture of comigrating rt-pcr fragments. supporting this contention is a prior report that showed hepg transcripts to undergo splicing of exon alone. based on its size alone, the 1l- transcript lacking exon could encode the smaller, immunoreactive species recently described in human sperm. based upon their sequences, conventional translational initiation events can not explain how human pn- or pt- derived transcripts are translated into stable shbg isoforms. the first potential aug start codon within exon 1n heads a short, four amino acid open reading frame. the subsequent aug codon lies at the beginning of a amino acid open reading frame; a termination codon precludes its merge into the exon shbg/abp reading frame. exon 1t is devoid of an aug start codon, the first potential aug start codon within exon of pt- derived transcripts occurs at the beginning of a short open reading frame consisting of only amino acids. for such transcripts, it has been suggested that translation may initiate within exon at a downstream aug codon that shares the shbg/abp open reading frame, or that they may utilize alternative, rare start codons within exon 1t. we note that, beginning with the cug at position + there are eight possible alternative cug, uug, gug, or aua start codons within the long exon 1t open reading frame that merges with the shbg/abp reading frame in exon more work is required to determine the biologic fates of transcripts originating from pt and pn. functionally, as has been seen with other genes, it is feasible that nontranslated transcripts originating from pt and pn could be regulatory in nature, affecting the expression of those that do encode shbg and stable shbg isoforms. the 1l- transcripts lacking exons and or exon alone could encode the smaller shbg species we detected in hepg lncap and mcf- cells. a similar sized species was not identified in affinity purified shbg from human plasma, hence, it might not bind steroids, and/or it may remain intracellular. the existence of an intracellular shbg species that does not bind steroid, but retains the rshbg membrane-binding motif within exon would be quite exciting, as it could potentially affect rshbg signaling, as well as other shbg mediated properties. in addition to our finding of a smaller shbg species in cell lines, human hypothalamus and cerebrospinal fluid have been shown to contain an immunoreactive shbg species which is -kd smaller than that of serum shbg. lower molecular weight species have been isolated from human male genital tissue and myocardium. taken together, these results suggest that a deeper investigation of alternatively spliced transcripts originating from pl may explain these findings. human shbg gene structure the existence of human shbg gene transcripts originating from pt and pn fits a larger picture in which the mammalian shbg gene is in a state of evolutionary flux,'hijacking' promoter elements from adjacent upstream genes on chromosome 17p that are transcribed in the opposite orientation. pt, which is located kb upstream of pl, shares regulatory elements with the adjacent polyamine n-acetyltransferase gene. based on chromosomal mapping of shbg exon 1n, pn is located within intron of the fxr gene. such overlapping transcriptional units and bi-directional promoter arrangements have been described for a number of genes; computational studies of gene organization predict them to occur frequently within the human genome. the rat possesses homologous exon 1n sequences, but these have not been reported in mature rat shbg transcripts. while prior work suggested that certain orthologous sequences might be incorporated into mature human transcripts, using race assays, we did not specifically detect the existence of a human homologue of rat pa. furthermore, the putative human exon 1a region contains no potential start codons, thus, transcripts originating from a human pa would run into the same functional and translational questions as do transcripts derived from pt. relative abundance of human shbg transcripts in tissues and cell lines transcripts derived from pl, pt, and pn are expressed differentially in normal prostate, breast, liver, and testis. the relative expression of the shbg/abp transcript is consistent with our hypothesis regarding local generation of shbg vs. the utilization of plasma shbg- that immediate, rapid effects of shbg, including those mediated by the rshbg signaling pathway, are likely to be regulated through local expression of shbg within responsive tissues; the concentration and/or availability of plasma shbg is not responsive to shbg needs in particular sites. each of the cell lines we examined had a reduced abundance of total 1l transcripts as compared to their corresponding normal tissues, and more specifically this was the case for the shbg/abp transcript. furthermore, the cancer cell lines examined in these studies had a greater overall abundance of alternatively spliced pl derived transcripts than did their corresponding normal tissues. if shbg gene expression is indeed modulated in prostate and breast cancer, this could alter their androgen and estrogen responsiveness.  in conclusion, our findings provide a broad and detailed picture of human shbg transcript structure and expression. at the same time, they also raise many new and important questions, including the viability of novel alternatively spliced transcripts, their functions in hormonally responsive tissues, including the breast and prostate, and the role that aberrant shbg gene expression may play in cancer. 
these results extend our understanding of human shbg gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant shbg gene expression may play in cancer. transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the lncap, mcf- and hepg cell lines. alternative splicing was more pronounced in the cancer cell lines. the shbg gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter, and two minor transcripts. initial findings reveal the existence of a smaller immunoreactive shbg species in lncap, mcf- and hepg cells. we report that transcriptional expression of the human shbg gene is far more complex than previously described. each expresses the shbg transcript, albeit in varying abundance.
 human sex hormone-binding globulin regulates free sex steroid concentrations in plasma and modulates rapid, membrane based steroid signaling. shbg is encoded by an eight exon-long transcript whose expression is regulated by a downstream promoter. the shbg gene was previously shown to express a second major transcript of unknown function, derived from an upstream promoter, and two minor transcripts.  we report that transcriptional expression of the human shbg gene is far more complex than previously described. pl and pt direct the expression of at least six independent transcripts each, resulting from alternative splicing of exons and/or we mapped two transcriptional start sites downstream of pl and pt, and present evidence for a third shbg gene promoter within the neighboring fxr gene; pn regulates the expression of at least seven independent shbg gene transcripts, each possessing a novel, 164-nt first exon. transcriptional expression patterns were generated for human prostate, breast, testis, liver, and brain, and the lncap, mcf- and hepg cell lines. each expresses the shbg transcript, albeit in varying abundance. alternative splicing was more pronounced in the cancer cell lines. pl- pt- and pn-derived transcripts were most abundant in liver, testis, and prostate, respectively. initial findings reveal the existence of a smaller immunoreactive shbg species in lncap, mcf- and hepg cells.  these results extend our understanding of human shbg gene transcription, and raise new and important questions regarding the role of novel alternatively spliced transcripts, their function in hormonally responsive tissues including the breast and prostate, and the role that aberrant shbg gene expression may play in cancer. 