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Departments of 1 Medicine2 Biochemistry, Division of Gastroenterology and Hepatology, Medical College of Wisconsin and Clement Zablocki VA Medical Center, Milwaukee, WI 53295, USA
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(Received 25 November 2003;
accepted after revision 13 January 2004;
first published online 14 January 2004)
Corresponding author B. Seetharam: VA Medical Center, Research 151, 5000 West National Avenue, Milwaukee, WI 53295, USA. Email: seethara{at}mcw.edu
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Introduction |
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Earlier studies have shown that TC is synthesized and secreted by different types of human cells (Seetharam & Li, 2000) and its mRNA is distributed widely in many human tissues (Li et al. 1994). Despite these studies very little is known about whether TC gene expression is regulated in tissues, particularly, intestine, and kidney, the two sites where its expression is physiologically important. Endogenous TC expressed by the intestinal epithelial cells is known to complex with Cbl liberated from gastric intrinsic factor following internalization of IF–Cbl from the apical side (Seetharam, 1999). However, the physiological role of endogenous TC synthesized in the renal proximal tubular epithelial cells (PTCs) is not known. It is likely that endogenous TC synthesized locally by PTCs (Ramanujam et al. 1991a) may aid in mediating the exit of reabsorbed Cbl back into the circulation (Ramanujam et al. 1990), particularly in rodents, where kidney is thought to be the storage site for Cbl (Scott et al. 1988; Birn et al. 2003).
The current studies were undertaken to address issues related to the regulation of expression of the TC gene in the rat intestine and kidney. The results of our study show that the TC gene is regulated in both rat tissues during postnatal development. In the intestine, the TC gene is regulated regionally along the length of intestine with high levels of expression in the jejunum and ileum relative to its levels in the duodenum. In the ileum TC mRNA levels are regulated in a biphasic manner with similar levels of sharp decline and increase of the transcript between days 4 and 12, and between days 12 and 24, respectively. In contrast, TC transcript levels in the kidney increased dramatically between days 4 and 12 followed by a sharp decline between days 16 and 24. In addition to different patterns of transcript expression levels in the intestine and kidney during postnatal development, intestinal but not kidney levels of TC mRNA were sensitive to adrenalectomy and cortisone treatment.
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Methods |
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Rat visceral yolk sac cDNA library constructed in the vector 
gt 11 was screened using 32P-labelled human TC cDNA and after tertiary screening, two positive clones were used for phage DNA isolation. The phage DNA isolated was digested with EcoR 1 and subjected to 1% agrose gel electrophoresis. Two cDNA inserts of 1.4 and 1.0 kb were identified. The two clones were then subcloned into the EcoR 1 site of bluescript-KS II plasmid vector (Stratagene). The purified plasmid DNA from the 1.4 kb insert was subjected to double strand sequencing by the dideoxy chain termination method (Sanger et al. 1977). Sequence alignment of rat TC and the published sequence of human, mouse and bovine TC were performed by Jellyfish (Lab Velocity) version 1.4 using matrix-Gonnet.
Experimental animals and tissue collection
All rats used in the study were from the Sprague-Dawley strain. Adrenalectomized rats, sham-operated rats and adult normal rats without any surgery were purchased from SASCO (Omaha, NE, USA). Adrenalectomy was performed on day 20. Two days after arrival, some adrenalectomized rats were treated orally with cortisone acetate (10 µg ml–1) added to drinking water that also contained 0.9 M NaCl, while others were allowed to drink normal saline. The feeding regime was followed for 6 days and serum cortisone levels determined by the competitive binding assay of Bassett & Hinks (1969), modified according to Henning (1978). The cortisone levels were 250, 0.1 and 185 ng ml–1 in sham-operated rats, adrenalectomized rats and adrenalectomized rats treated with cortisone acetate, respectively. Six rats were used in each of these three groups. Four male adult (24 days) rats were used for collecting the intestinal tissue which was then divided into duodenum, jejunum and ileum. Terminally pregnant rats that were housed in individual cages were checked on due date every 3–4 h and when the pups were delivered they were labelled as 0 days old. The pups were allowed to stay with the mother and were killed on day 4, 8, 12, 16 and 24 to harvest the kidney and intestine. Prior to harvesting the kidney and intestine, some animals were perfused with phosphate-buffered saline through the left ventricle of the heart as described earlier (Ramanujam et al. 1990). In some instances, the intestine and kidneys were harvested from pregnant rats of 19 day gestation and lactating mothers following the shifting of the pups to suckle with surrogate mothers. All adult and pregnant rats were anaesthetized with sodium phenobarbital (10 mg kg–1) administered intravenously. Pups (4–24 days old) were killed by decapitation to collect the intestine and kidney.
These procedures were approved by the Medical College of Wisconsin animal research committee, a member of the Association for Assessment and Accreditation of Laboratory Animal Care.
All the tissues harvested, except the intestine, were frozen in liquid nitrogen after removal. Intestinal tissue was flushed with ice-cold saline before being frozen in liquid nitrogen.
mRNA isolation and Northern blotting
Total mucosal RNA from either the entire adult male rat intestine or from its anatomical regions, yolk sac (from pregnant rats of 14 days gestation), kidney and intestine of both male and female rats of different ages was isolated using RNAzol (Tel-Test, Inc, TX, USA) and mRNA was isolated by the PolyATtract isolation system (Promega, WI, USA) as described by the manufacturer. Northern blots were generated using 2 µg of mRNA isolated from the intestine and yolk sac, and in the case of other rat tissues a commercial blot (Clonetech, Paloalto, CA, USA) containing 2 µg mRNA was used. The two probes used, rat TC cDNA and mouse ß-actin cDNA were 32P-labelled using the random primer method of Feinberg & Vogelstein (1983). The blots were probed with 32P-labelled rat TC cDNA [specific radioactivity 
1 x 109 d.p.m. (µg DNA)–1] using the same hybridization conditions described earlier (Li et al. 1994). In order to correct for the possible variation of mRNA loaded to each lane and to make sure of the integrity of the mRNA samples used, the same blots were stripped and rehybridized with the full length (2 kb) 32P-labelled mouse ß-actin cDNA (specific radioactivity 1 x 109 d.p.m. (µg DNA)–1). The autoradiographs were visualized for TC mRNA after exposure in the presence of an intensifying screen for 5 days. For visualization of ß-actin mRNA, the blots were exposed in the absence of an intensifying screen. Multiple exposure times were carried out to ensure that the signals were in the linear range of the film sensitivity.
Quantitative analysis
Signals of interest from the Northern blots were quantified by scanning with a response spectrophotometer (Jolstens Graphic products, Chicago, IL, USA). The ratios of image intensities of TC and actin mRNA were calculated and expressed as such. In some experiments, the ratio is expressed as a percentage. For graphical presentation, values for individual animals from each experimental group were calculated as mean ±S.E.M. The number of animals in each group is given in the figure legends. Statistical significance was assessed by one-way or two-way ANOVA. A P value < 0.05 was considered significant.
Ileal mucosal uptake of IF–Cbl and formation of TC–Cbl complex
In order to determine whether adrenalectomy had any direct effect on TC synthesis and ileal formation of the TC–Cbl complex, an essential step before absorbed Cbl enters the circulation, the following experimental setup was used. Rat IF–[57Co]Cbl (0.74 pmol, 400 000 d.p.m.) was orally administered to normal, adrenalectomized, and cortisone-treated adrenalectomized rats. Two hours later, the animals were killed and their intestines washed with ice-cold saline. The ileal segments containing a sufficient amount of radioactivity were selected and exposed to 10 ml of 2.5 mM EDTA (pH 6.0) for 15 min at 22°C in a small Petri dish to remove surface-bound radioactivity. The segments were then equilibrated for 10 min in phosphate-buffered saline. The radioactivity in the post-washed segments was considered to be internalized [57Co]Cbl as washing with EDTA (pH 6.0) removed only surface-bound radioactivity. A 10% homogenate was prepared in phosphate-buffered saline containing 1 mM phenylmethylsulphonylfluoride, using mucosa scraped with a glass slide. The internalized radioactivity in the homogenates was extracted with 0.5% Triton X-100 for 18 h at 5°C. The homogenate thus treated was centrifuged for 1 h at 100 000 g. The supernatant containing > 90% of total internalized counts was used to study the distribution of [57Co]Cbl between free IF or IF still bound to the IF–Cbl receptor, or to haptocorrins (HCs) or TC by immunoprecipitation using antisera to rat IF, rat IF–Cbl receptor, porcine haptocorrins (HC), or human TC as described before (Ramasamy et al. 1989). The ability of porcine HC antiserum to recognize HCs from other species, including rat, has been established before (Lee et al. 1989).
[57Co]Cbl binding capacity
[57Co]Cbl (ICN Radiochemicals, Irvine, CA, USA; specific activity 1.3 µCi µg–1) binding to either a medium of cultured renal or intestinal segments or to plasma prepared from 21-day-old non-pregnant female and pregnant dams, or 25-day-old lactating mothers, was determined by the charcoal absorption method (Gottlieb et al. 1965). Ileal segments, longitudinally opened and washed (2 cm long), or kidney pieces (200 mg), from different ages were cultured in Dulbecco's minimum essential medium in the absence of bovine serum and cobalamin for 4 h. After the initial binding of [57Co]Cbl to plasma, the protein-bound radioactivity (100 000 d.p.m.) was immunoprecipitated with antiserum to human TC raised in New Zealand White rabbits (Ramanujam et al. 1991b) or normal preimmune rabbit serum (25 µl) and 125 µl of a 1: 1 suspension of protein A–sepharose. The radioactivity precipitated with preimmune serum (3–5%) was subtracted from that precipitated with TC antiserum and was taken as the amount of Cbl bound to plasma TC.
The validity of the use of human TC antiserum to immunoprecipitate Cbl complexed to either rat serum or culture medium from rat tissue explants was confirmed as follows: rat serum-bound [57Co]Cbl (1 ml, 50 000 d.p.m.), prepared using the charcoal separation method (Gottlieb et al. 1965), was incubated with undiluted human TC antiserum (2.5–25 µl) for 18 h at 5°C and then 100 µl of protein A–sepharose suspension (1: 1) was added and further incubated overnight. The radioactivity associated with the beads was determined following centrifugation of the mixture and was taken as that bound to TC. In addition, immunoblotting using human TC antiserum was carried out using recombinant human TC (Kalra et al. 2003) or 5 µl of 5-fold-concentrated rat serum. Prior to blotting, the proteins were separated on non-reducing SDS-PAGE (7.5%) and immunoblotting for TC was carried out using 5000-fold diluted preimmune or anti-human TC serum as described before (Yammani et al. 2003).
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Results |
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To obtain a cDNA probe that can be efficiently used for the measurement of TC mRNA levels in the rat tissues, we isolated a full length TC cDNA from a rat visceral yolk sac library using a full length human TC cDNA clone (Li et al. 1993) as a probe. Two clones were obtained, each with an insert size of 1.2 kb. Initial sequencing confirmed the identity of both clones by comparison to published sequences of human (Li et al. 1993), bovine (Fedesov et al. 1999) and mouse (Hasegawa & Foote, 1998) TC. Amino acid sequence comparison (Fig. 1) of the mature rat TC with mouse, human and bovine TC revealed an overall 83% identity. The secondary structure predictions and hydropathy index of rat TC were very similar to human TC (Li et al. 1993) (data not shown). Rat TC sequence revealed the presence of six cysteine residues in identical positions as in mouse, human and bovine TC (Kalra et al. 2003) or other Cbl-binding proteins (Li et al. 1993) and the presence of only one potential N-glycosylation site at the same position as in TC from all the three species, 33NPS35. However, this site is not used in rat TC for N-glycosylation due to the presence of a proline residue which inhibits N-glycosylation at this site (Kaplan et al. 1987).
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Northern blot analysis using mRNA from several rat tissues revealed a single transcript of 1.9 kb (Fig. 2, upper panel), but the transcript levels in these tissues varied. Normalization with cytoskeletal ß-actin mRNA (Fig. 2, lower panel) and using the transcript level in the kidney as 100%, the transcript levels in the liver, lung, yolk sac and intestine were 89%, 84%, 60% and 55%, respectively. In contrast to these tissues with relatively high levels of TC transcript expression, its levels in heart, brain, spleen and skeletal muscle were 35%, 30%, 25% and 24%, respectively. When standardized with respect to muscle ß-actin, the TC mRNA levels in all the tissues followed a similar pattern of distribution as above, except in heart and muscle, where they were actually lower due to a much higher expression of this form of ß-actin. These results indicated that the TC gene is expressed in many tissues in rat, but due to its role in Cbl transport to the circulation in the intestine and its potential role in the mobilization of stored Cbl from the kidney, we focused our attention on examining various physiological conditions that could affect TC gene expression in these two tissues.
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Since some of the studies on TC protein expression in rat necessitated the use of human TC antiserum, we wanted to initially test whether human and rat TC are immunologically identical. When human TC antiserum was added in increasing amounts (2.5–25 µl), it was able to immunoprecipitate the labelled Cbl bound to rat serum and 100% of the label was immunoprecipitated with 25 µl of human TC antiserum (Fig. 3, left panel). This result indicated that in rat serum almost all of the unsaturated Cbl binding is due to TC and that human TC antiserum is able to immunoprecipitate rat TC. In addition, immunoblot analysis (right panel) revealed that human TC antiserum is able to recognize rat TC protein as a single protein band of 43 kDa in the serum (lane 3) as well as recombinant human TC (lane 2). This band was absent in rat serum when the immnoblot was carried out with preimmune rabbit serum (lane 1).
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Due to its role in transcellular transport of dietary and biliary Cbl in the intestine, we examined the ontogeny of TC gene expression in the intestine of rats aged between 4 and 24 days after birth. During this time frame of postnatal development, changes occur in the intestine when the source of Cbl changes from mother's milk to a solid diet and the absorption mechanism changes from IF-independent (HC-dependent) to IF-dependent (Seetharam, 1994).
In this context, we first wanted to examine whether TC gene expression occurs along the entire length of the small intestine of an adult rat or whether its expression is confined to distal regions of the gut where an IF-dependent mechanism of Cbl uptake occurs. Our initial assay of TC transcript levels along the duodenal–ileal axis of an adult rat (Fig. 4, upper panel) revealed similar high levels of TC transcript in jejunum and ileum relative to its levels in the duodenum where, based on normalization with ß-actin, the TC transcript levels were only 25% (Fig. 4, lower panel). Although both ileum and jejunum expressed similar high levels of TC transcript consistent with a more generalized distribution of Cbl absorption and transport machinery in the rat, we selected ileum to study factors that might regulate TC mRNA expression in the intestine.
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Since the protein or mRNA changes of many functional proteins in the intestine during postnatal development in the rat are attributed to changes in plasma glucocorticoid levels, we wanted to examine whether cortisone has any effect on TC mRNA levels in this tissue. Furthermore, since kidney exhibited a different pattern of TC mRNA expression during postnatal development, we hypothesized that cortisone may not have any role in TC mRNA expression in the kidney. In order to examine these possibilities, we determined TC mRNA levels in these two tissues from adult rats subjected to adrenalectomy.
Relative to sham-operated control rats (Fig. 6, upper left panel, lane 1) TC mRNA levels demonstrated a sharp decline (lane 2) in the ileum of adrenalectomized rats and treatment of these rats with cortisone for 6 days restored the transcript to normal levels (lane 3). Normalization of ileal TC mRNA levels with ß-actin transcript levels demonstrated (Fig. 6, lower left panel) a 4-fold (lane 2) decline in transcript levels in adrenalectomized rats relative to sham-operated control rats (lane 1). The transcript levels returned to 85% of the normal values following a 6 day treatment of adrenalectomized rats with cortisone (lane 3). In contrast to ileum, TC mRNA levels in the kidney of adrenalectomized rats (right panels) before (lane 2) and after (lane 3) treatment with cortisone were not significantly altered relative to its levels in the kidney of sham-operated rats (lane 1). Normalization with ß-actin transcript levels (lower panel) revealed that, relative to control rat kidney (lane 1), there were insignificant changes in the transcript levels in adrenalectomized rats before (lane 2) or after (lane 3) cortisone treatment. These observations, along with the noted changes in transcript levels in the kidney and ileum during postnatal development, suggested that cortisone plays a crucial role in regulating TC transcript levels in the ileum while it has no significant effect on TC mRNA levels in the kidney. In order to test whether decreased levels of TC mRNA transcript in the ileum of adrenalectomized rats also resulted in decreased amounts of TC protein in the ileal mucosa, the amount of labelled Cbl transferred from IF to TC in the mucosal extracts was measured 2 h following the oral administration of IF–[57Co]Cbl.
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After oral administration of IF–[57Co]Cbl, the distribution of internalized [57Co]Cbl bound to various proteins involved in absorption and transport of Cbl was determined (Table 1). In 2 h, a time frame in which the [57Co]Cbl taken up by the ileum will be still within the mucosal cells, there was an almost equal distribution (45–50%) of the labelled Cbl bound to IF or TC with little or no Cbl on HC. However, in adrenalectomized rats, the amount of Cbl bound to TC declined by almost one-half with an increase in the amount of free Cbl (from 5 to 20%) and no change in the amount of Cbl bound to either free IF or IF still complexed with the IF–Cbl receptor. Treatment of adrenalectomized rats with cortisone resulted in an increase in the amount of Cbl that was bound to TC. These observations clearly indicated that adrenalectomy resulted in the decreased association of internalized Cbl with endogenous TC, consistent with decreases in its transcript and protein levels.
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Another physiological condition which might influence TC mRNA expression in rats is pregnancy. During pregnancy, there is enhanced gastrointestinal absorption of Cbl (Robertson & Gallagher, 1983) and Cbl balance is tilted towards the fetus (Graber et al. 1971; Fernandes-Costa & Metz, 1979). However, it is not known whether these changes in Cbl balance during pregnancy are accompanied by increased TC mRNA and protein expression in the maternal tissues and thus raising the plasma TC levels in the maternal circulation. In order to explore these possibilities, TC mRNA levels in the maternal kidney and ileum and plasma TC levels in age-matched non-pregnant female rats, pregnant rats of 21 days gestation and lactating mothers were measured.
Plasma [57Co]Cbl binding capacity due to TC (Fig. 7A) in age-matched non-pregnant female control rats, pregnant rats of 21 days of gestation, and lactating mothers, were between 1.2 and 1.4 pmol ml–1 indicating that TC activity levels are not altered during pregnancy (column 1) or in lactating mothers (column 2) relative to non-pregnant females (column 3). In addition, TC mRNA levels in the ileum (Fig. 7B) and kidney (Fig. 7C) did not show any significant changes in transcript levels in age-matched non-pregnant female control rats (lane 1), lactating mothers (lane 2), or pregnant mothers (lane 3), indicating that TC levels are not regulated in the tissues of pregnant mothers.
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Discussion |
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The rat TC sequence, like human (Li et al. 1993) and mouse (Hasegawa & Foote, 1998) TC contains eight cysteine residues at identical positions (Fig. 1, stars) while bovine TC (Fedesov et al. 1999) contains only six cysteine residues. The cysteine residues at positions 65 and 78 in bovine TC are replaced by serine residues. Recent studies have shown that both human (Kalra et al. 2003) and bovine TC (Fedesov et al. 1999) form three intramolecular disulphide bonds using the six conserved residues, two of which, C147–C187 and C98–C291 in human TC (Kalra et al. 2003) are important to its function and intracellular stability, while the other disulphide bond formed between C3 and C249 has no role in either Cbl binding or stability of TC. Earlier sequence-comparison studies (Seetharam & Li, 2000) have shown that, like TC, other mammalian Cbl binding proteins, gastric intrinsic factor and haptocorrins also contain six conserved cysteine residues at the same position. Taken together, these studies suggest that, like human and bovine TC, both IF and HC may also form similar intramolecular disulphide bonds needed to acquire the folding that is essential for their common function: high affinity Cbl binding.
The physiological significance of the observations noted in this study, the wide tissue distribution, different patterns of TC mRNA expression in kidney and intestine during postnatal development, and differential effects of cortisone on TC gene expression in the rat kidney and intestine, is not fully understood. However, these observations raise some interesting questions regarding the role of TC at these sites. TC is constitutively secreted from cells in culture (Seetharam & Li, 2000, and references therein), with or without Cbl being bound to it, depending upon the availability of an excess of intracellular Cbl in the free form. The finding of varying levels of TC mRNA in rat tissues may simply reflect the varying amounts of Cbl mobilized from these sites to enter the circulation for either excretion or tissue exchange. It will be interesting to investigate further whether TC expression in cells is regulated by the flux of Cbl that traverses their plasma membranes and whether its levels are drastically altered in intracellular Cbl deficiency. Our attempts to quantify TC mRNA distribution among various cell types, particularly in the liver, kidney and intestine, were not successful due to the extremely low abundance of the transcript in these tissues. However, it is important to note that the primary cells that synthesize and secrete TC in the kidney and intestine are the epithelial cells.
Within the rat intestine, unlike in humans (Hagedorn & Alpers, 1977), IF–Cbl uptake is not exclusively limited to terminal ileum and it occurs in the distal parts of the jejunum as well (Seetharam et al. 1992). Since endogenous TC synthesized by the absorptive epithelial cells of the intestine plays a key role in the final stages of transcellular transport of Cbl (Seetharam, 1994), it is not surprising to find similar high levels of expression of the TC gene in both rat jejunum and ileum. Previously (Ramasamy et al. 1989) we have shown that TC synthesized during the early suckling period is physiologically active in mediating transcellular transport of Cbl through the ileal cells of suckling rats, independent of the mechanism of its uptake (IF-dependent or -independent). Although it is not clear how Cbl present in the rat milk (Raaberg et al. 1989) is taken up during the suckling period, it is likely that it occurs bound to haptocorrin and in support of this suggestion there are studies that have shown the binding of HC–Cbl to the apical membranes of nenonatal intestine (Hu et al. 1991). It is interesting that the increases in TC transcript and protein levels in the ileum of rats between the age of 12 and 24 days coincide with the increases in the production of IF by the gastric mucosa (Dieckgraefe et al. 1988) and of its receptor (Ramasamy et al. 1989) in the ileum, suggesting that during this phase of ileal development, there could be a coordinated regulation of these three proteins to facilitate the transcellular transport of Cbl by the IF-mediated Cbl transport system.
It is well known that the changes in the transcript levels, particularly in the intestine, of several genes during the postnatal developmental of the rat are influenced by glucocorticoids (Henning et al. 1994) whose levels start to increase by day 14 and reach peak levels by day 24 (Henning, 1978). Viewed in this context, it is likely that the increase in TC mRNA transcript levels in the ileum from days 12 to 20 is due to rising plasma cortisone levels. This suggestion is directly supported by the noted decreases in TC transcript levels and the mucosal formation of the TC–Cbl complex in adrenalectomized rats and their return to normal levels following cortisone treatment. Previous studies have shown that adrenalectomy in adult rats also drastically reduces TC receptor levels and the plasma transport of absorbed Cbl to the kidney (Bose et al. 1995). In addition, gastric IF mRNA (Dieckgraefe et al. 1988) levels also decline in adrenalectomized rats. Although decreased IF levels will not be a factor in the current studies, it is difficult to assess how much the decrease in mucosal formation of TC–Cbl contributes to decreased plasma transport to peripheral tissues. Although the rise in TC mRNA levels from age 12 to 24 days may be due to rising levels of cortisone, it is not clear why TC transcript levels in the ileum are high on day 4 and then have declined by nearly 50% by day 12. One possible explanation for this observation is that some unique factor/s, including high levels of cortisone in the fetal circulation that is noted in full term human fetuses (Murphy, 1982), might still temporarily influence TC transcript levels at birth and a few days later also in rat and then cease to play a role during the early weaning period. Decline of TC transcript levels between days 4 and 12 support this possibility and further studies are needed to understand the molecular mechanism/s involved in these processes.
Rat kidney had the highest levels of the TC transcript and during postnatal development peak levels of the transcript were reached during the suckling period from days 4 to 16. The physiological role of TC synthesized by the proximal tubular epithelial cells of the kidney is not fully understood. However, it has been suggested that it could play a role in the release of reabsorbed and stored Cbl back into the circulation. There is some evidence in humans with renal carcinoma (Jensen et al. 1983) or renal insufficiency (Areekul et al. 1993) that plasma TC levels are elevated in these disorders. However, the elevation of plasma TC levels is thought to be due to reduced blood flow and filtration rate that results in reduced reabsorption of TC–Cbl. In addition, patients with end-stage renal disease have elevated levels of plasma homocysteine (Hyndman et al. 2003) suggesting that Cbl deficiency, one of the causes of increased homocysteine levels, may also occur due to loss of tubular reabsorption of plasma TC–Cbl. Taken together, these studies have suggested that the kidney plays an important role in both Cbl homeostasis and maintaining plasma TC levels, but additional studies are required to determine the role of endogenous TC in these processes.
The reabsorption of filtered Cbl bound to TC appears to develop early in rats and it is suggested, based on studies of phosphate reabsorption, that the reabsorption capacity of the kidney develops very early (Haramati et al. 1988), and one of the factors that influences the reabsorption capacity of a particular nutrient is its concentration in the mother's milk and the requirement of that nutrient for growth during postnatal development (Traebert et al. 1999). Thus, it is not surprising that reabsorption of Cbl, an important nutrient whose levels in rat milk are high (Raaberg et al. 1989), also develops early. In support of these suggestions are studies that have shown unaltered high levels of TC receptor during postnatal development of rat kidney (data not shown). Mobilization of filtered Cbl bound to endogenous TC may provide Cbl to other organs in suckling rats during their transition from a liquid to a solid diet, which occurs around 19–20 days after birth.
In contrast to its effect on ileal TC mRNA transcript levels, cortisone appears to have no significant effect on the TC transcript levels in the kidney. This observation is supported by the finding of steady state increases in transcript levels from days 4 to 12 when cortisone levels are low, followed by their leveling off between days 12 and 16, and a sharp decline from day 16 to day 24, a time frame during which cortisone levels are peaking and reach maximum levels, respectively. Thus, it is likely that TC mRNA expressed in the kidney is not sensitive to regulation by cortisone. It is interesting to note that in rabbit, mRNA encoding trehalase (Galand et al. 1995), a brush border membrane protein which is expressed in both intestine and kidney, is similarly modulated in these two tissues during postnatal development as well as by cortisone.
The lack of up-regulation of TC mRNA levels in maternal ileum and kidney in pregnant mothers and the unaltered levels of plasma TC activity during pregnancy in rats suggests that the rate-limiting step of Cbl delivery to the fetus may be controlled at the maternal–fetal interface, where free Cbl is known to accumulate (Graber et al. 1971; Fernandes-Costa & Metz, 1979). The finding of high levels of TC mRNA in the yolk sac placenta suggests that TC synthesis at this site may be important for delivery of Cbl to the fetus. In support of this suggestion are earlier studies (Polliotti et al. 1997) that have shown the presence of an endocytic mechanism for the uptake of TC–Cbl in the near-term rat visceral yolk sac. In contrast to rats, studies in humans using the perfusion technique have shown that Cbl transport across the human placenta as well as in the fetal circulation may be promoted by haptocorrins (Perez-D'Gregorio & Miller, 1998). Thus, it appears that the placental transport of maternal Cbl to the fetus is a complex event and that the physiological role of yolk sac-placental expression of TC, if any, in Cbl transport to the fetus needs further studies.
The molecular mechanism/s involved in the developmental and hormonal regulation of the same gene expressed in different tissues is not fully understood and many factors need to be considered. These include: varying levels of cytosolic glucocorticoid receptors during postnatal development and their sensitivity in mediating events; and different pathways selected, direct or circuitous, to influence gene transcription and changing levels of specific transcription factors that affect gene transcription (Henning et al. 1994). In this regard, it is important to note, that although TC is the product of a housekeeping gene (Li et al. 1995), its expression in vitro is controlled by the relative ratios of expression of transcription factors Sp1 and Sp3 (Li et al. 1998) and by upstream stimulatory factor 1 (Li & Seetharam, 1998). Further in vivo studies are needed to examine whether the levels of these transcription factors are altered in vivo and directly affect transcription of the TC gene.
In summary, the results of this study show that in the rat TC, transcript levels are regulated in (a) a tissue-specific manner, (b) the ileum and kidney during their postnatal development, and (c) the ileum by cortisone. Further studies are required to understand the molecular mechanism/s involved in TC regulation which in turn may help to understand better the relationship between TC expression and flux of Cbl transported across the plasma membranes of different tissues, particularly absorptive epithelial cells of the intestine and kidney.
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References |
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) of the culture medium of intestinal and renal explants are shown on the right. The values in the right-hand panels are mean ±S.E.M. from five different animals in each age group. Other details are provided in Methods.
