A critical role for enhanced TGF-α and EGFR expression in the initiation of parathyroid hyperplasia in experimental kidney disease

Mario Cozzolino, Yan Lu, Tetsuhiko Sato, Jing Yang, Ignacio Gonzalez Suarez, Diego Brancaccio, Eduardo Slatopolsky, Adriana S. Dusso

Abstract

The parathyroid hyperplasia secondary to kidney disease is associated with enhanced expression of the growth promoter transforming growth factor-α (TGF-α). TGF-α stimulates growth through activation of its receptor, the epidermal growth factor receptor (EGFR), normally expressed in the parathyroid glands. Because enhanced coexpression of TGF-α and EGFR causes aggressive cellular growth, these studies utilized highly specific inhibitors of EGFR tyrosine kinase, a step mandatory for TGF-α-induced EGFR activation, to assess the contribution of growth signals from enhanced expression of TGF-α exclusively or both TGF-α and EGFR to the rapid parathyroid growth induced by kidney disease and exacerbated by high-phosphorus (P) and low-calcium (Ca) diets in rats. The enhancement in parathyroid gland weight and proliferating activity (proliferating cell nuclear antigen/Ki67) induced by kidney disease and aggravated by either high P or low Ca intake, within the first week after 5/6 nephrectomy, in rats, coincided with simultaneous increases (2- to 3-fold) in TGF-α and EGFR content. Conversely, prevention of the increases in both TGF-α and EGFR paralleled the efficacy of either P restriction or high-Ca intake in ameliorating uremia-induced parathyroid hyperplasia. More importantly, suppression of TGF-α/EGFR signaling, through prophylactic administration of potent and highly selective inhibitors of ligand-induced EGFR activation, completely prevented both high-P- and low-Ca-induced parathyroid hyperplasia as well as TGF-α self-upregulation. Thus enhanced parathyroid TGF-α/EGFR expression, self-upregulation, and growth signals occur early in kidney disease, are aggravated by low-Ca and high-P intake, and constitute the main pathogenic mechanism of the severity of parathyroid hyperplasia.

  • secondary hyperparathyroidism
  • growth arrest
  • epidermal growth factor receptor
  • tyrosine kinase inhibitor
  • renal failure

in chronic kidney disease, elevated serum levels of parathyroid hormone (PTH) cause osteitis fibrosa, bone loss, and cardiovascular complications, all of which contribute significantly to increased morbidity and mortality (8, 13, 14). Hypocalcemia, hyperphosphatemia, and 1,25-dihydroxyvitamin D (calcitriol) deficiency are the main direct causes of parathyroid cell growth and increased PTH synthesis and secretion (10, 17, 24). Parathyroid cell proliferation rather than hypertrophy is the main determinant of parathyroid gland enlargement and, consequently, high serum PTH levels (18, 23, 25).

The lack of an appropriate parathyroid cell line as well as the rapid dedifferentiation of normal and hyperplastic parathyroid cells in culture (4) have impeded the identification of the mechanisms underlying the switch of a quiescent parathyroid cell to one that divides in response to kidney disease, hypocalcemia, hyperphosphatemia, or calcitriol deficiency. Therefore, at present, experimental models of kidney disease constitute the only approach for further characterization of the pathogenesis of parathyroid hyperplasia. Using the 5/6 nephrectomized rat model, we have previously found that, similar to the enhanced TGF-α expression of hyperplastic and adenomatous human parathyroid glands (12), elevations in parathyroid content of TGF-α associate with the hyperplastic growth, already present by day 7 after the onset of renal failure (9, 11). Not only was TGF-α expression higher in the hyperplastic parathyroid glands of 5/6 nephrectomized rats than in normal controls, but a high phosphorus (P) intake further enhanced parathyroid TGF-α levels and proliferating activity by day 7 after the onset of kidney disease (11), thereby doubling parathyroid gland size (10). In contrast, the efficacy of P restriction, high dietary calcium (Ca), or prophylactic calcitriol (or analog) therapy to counteract the parathyroid cell growth induced by the onset of kidney disease was associated with prevention of uremia-induced increases in parathyroid TGF-α (9, 11). The potential role of TGF-α in the parathyroid hyperplasia induced by low dietary Ca has not been addressed.

Growth signals from enhanced parathyroid TGF-α require activation of the TGF-α receptor, the epidermal growth factor receptor (EGFR), a 170-kDa membrane glycoprotein with intrinsic tyrosine kinase activity (28), normally expressed in the parathyroid glands (12, 22). In several human carcinomas (1) as well as in nonneoplasic hyperproliferative disorders (26), enhanced coexpression of TGF-α and EGFR in a cell type results in an autocrine growth loop, which furthers TGF-α self-upregulation and, consequently, more aggressive cellular growth. In human parathyroid glands from primary adenomas or renal hyperparathyroidism, conflicting reports exist as to whether EGFR levels are enhanced in parallel with the increases in TGF-α content or maintained within the normal range (12, 22, 28). The goals of the present studies were to characterize 1) whether the rapid and potent mitogenic signals triggered by kidney disease and worsened by high dietary P within the first week after 5/6 nephrectomy resulted from exclusive upregulation of TGF-α or involved simultaneous increases in TGF-α and EGFR expression; 2) the potential role of the TGF-α/EGFR growth loop to low Ca-induced parathyroid hyperplasia in early kidney disease; and 3) the actual contribution of enhanced TGF-α/EGFR growth signals to parathyroid hyperplasia in experimental kidney disease. For the latter, we used potent and highly specific inhibition of TGF-α/EGFR-driven growth.

TGF-α/EGFR-driven growth involves TGF-α binding to the EGFR, which in turn induces EGFR dimerization and tyrosine phosphorylation of the EGFR by an intrinsic EGFR tyrosine kinase. This step, mandatory for the onset of downstream growth signals, is specifically inhibited by EGFR tyrosine kinase inhibitors. Two small-molecule EGFR tyrosine kinase inhibitors, the tyrphostin AG-1478 [4-(3-chloroanilino)-6, 7-dimetoxyquinazoline] and the quinazolinamine OSI-774 (Erlotinib) were used. Both molecules are highly selective for EGFR tyrosine kinase (2, 19), compete with ATP binding, and reversibly inhibit EGFR tyrosine trans-phosphorylation, thereby blocking the potent growth signals downstream from TGF-α binding to the EGFR in vitro (5) and in vivo (1, 19, 29). Prophylactic administration of AG-1478 or Erlotinib to 5/6 nephrectomized rats fed either high-P or low-Ca diets for 1 wk measured the efficacy of direct inhibition of EGFR tyrosine phosphorylation and, therefore, growth signals from the enhanced parathyroid TGF-α levels in ameliorating the severity of parathyroid hyperplasia.

These studies provide the first demonstration that growth signals from enhanced parathyroid expression of TGF-α and EGFR constitute a main pathogenic mechanism of the parathyroid hyperplasia that develops early after the onset of kidney disease and is aggravated by both low dietary Ca and high P intake.

METHODS

Experimental Protocols

Female Sprague-Dawley rats, 5–6 wk old, weighing 200–225 g, were subjected to 5/6 nephrectomy. Briefly, in a single surgical procedure, several branches of the left renal artery were ligated and the right kidney excised.

Protocol I: temporal association between parathyroid TGF-α and EGFR levels and proliferation rates.

The degree of parathyroid hyperplasia induced by the onset of kidney disease through 5/6 nephrectomy was modulated through dietary Ca and P manipulations. To further enhance the rate of parathyroid cell growth over that triggered by kidney disease, uremic (5/6 nephrectomized; U) rats were fed either a high-P (HP; 0.9% P; 0.6% Ca) (9) or a low-Ca diet (LCa; 0.6% P; 0.1% Ca) for 7 days after the onset of kidney disease. To counteract the increases in parathyroid cell proliferation induced by 5/6 nephrectomy, uremic rats were fed either a low-P (LP; 0.2% P; 0.5% Ca) or a high-Ca diet (HCa; 1.25% P; 2.0% Ca). All diets were purchased from Dyets (Bethlehem, PA). To control for potential changes in parathyroid cell proliferation induced by high dietary P independently of kidney disease, a group of normal rats was fed the high-P diet. Note that for the experimental groups with dietary P manipulations, only the quantification of parathyroid EGFR levels is presented in this manuscript.

Protocol II: efficacy of EGFR-tyrosine kinase inhibitors in suppressing high P- and low Ca-induced parathyroid hyperplasia in early experimental uremia.

protocol iia: high dietary p with intermittent ag-1478 dosing.

Female 5/6 nephrectomized Sprague-Dawley rats, 2 to 3 wk old, weighing 150–175 g, were fed a high-P diet (1.2% P; 0.4% Ca) and treated with DMSO as vehicle (V; U-HP+DMSO) or the small-molecule EGFR tyrosine kinase inhibitor AG-1478, 25 mg/kg body wt every other day (U-HP+AG-1478). AG-1478 (purchased from Calbiochem, San Diego, CA) was administered intraperitoneally (ip) in 1 ml of DMSO. The dose of AG-1478 is half of that effective to arrest EGFR-driven growth in aggressively growing tumors in mice when given daily (5) and was further adjusted considering a mouse:rat-metabolic ratio of 2:1. Because P restriction prevents the parathyroid cell growth induced by kidney disease within the first week after 5/6 nephrectomy, an additional group of 5/6 nephrectomized rats that were fed a low-P diet (0.2% P; 0.5% Ca) and treated with DMSO as vehicle (U-LP+DMSO) served as a negative control for parathyroid hyperplasia.

protocol iib: high-p/low-ca diets with daily erlotinib dosing.

Due to the reversibility of tyrphostin EGFR binding, similar studies were also conducted with daily administration of OSI-774 (Erlotinib, kindly provided by Genentech), at ip doses of 3 and 6 mg/kg body wt in 200 μl of DMSO to 5/6 nephrectomized rats fed either a high-P (1.2% P) or low-Ca (0.1% Ca) diet. The switch from AG-1478 to Erlotinib for daily dosing was based on the tendency for phosphate retention (despite the reduction of PTH) observed in the group receiving intermittent AG-1478. Significant increase in serum P with daily AG-1478 would compromise result interpretation. The criterion for dose selection with daily Erlotinib was similar to that indicated for AG-1478 and based on the pharmacology reported for OSI-774 for tumor suppression in mice (19). Two independent experiments with at least seven rats per experimental group were conducted for each dietary manipulation.

For all protocols, rats were killed after a 7-day treatment. At death, blood was drawn for analytic determinations. Parathyroid glands were surgically removed and weighed on a CAHN-31 microbalance (Cahn Instruments, Cerritos, CA). For immunohistochemistry, parathyroid glands were kept in 10% formaldehyde overnight and then transferred to 70% ethanol in water before mounting.

Experimental protocols were approved by the Animal Study Committee at Washington University School of Medicine.

Analytic Determinations

Plasma P and creatinine levels were determined using an autoanalyzer (COBAS-MIRA Plus, Branchburg, NJ). Ionized calcium (ICa) was measured using an ICa-specific electrode (model ICA-1; Radiometer, Copenhagen, Denmark). Intact PTH levels were measured by an immunoradiometric assay, specific for intact rat PTH (Immutopics, San Clemente, CA).

Immunohistochemistry

Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), Ki67, and TGF-α was performed on sections of formalin-fixed, paraffin-embedded parathyroid glands following the protocols described in previous studies for each specific protein (9). In the two independent experiments conducted for protocol IIa, PCNA quantification was normalized as follows: for each experiment, the average number of PCNA-positive nuclei per gland area was calculated in the P-restricted uremic rats. The % increases in proliferating activity were measured by the ratio between individual values for PCNA/area and the average PCNA/area in the respective LP group.

Immunohistochemical staining for EGFR was also performed on formalin-fixed, paraffin-embedded parathyroid glands using rabbit monoclonal anti-EGFR antibody (SC-03, Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody and a commercial immunohistochemical staining kit (Histostain-Plus, mouse; Zymed Laboratories). The specificity of the primary antibodies for immunohistochemical staining was tested in rat parathyroid tissue by replacing the primary antibody with mouse IgG1. Briefly, sections of rat parathyroid tissue were deparaffinized, rehydrated, and endogenous peroxidase quenched using 0.6% hydrogen peroxide in methanol. For EGFR immunostaining, tissue sections were microwaved in 10 mM citric acid buffer, pH 6.0, for 10 min. Tissue was then blocked with 10% preimmune goat serum and incubated with primary antibody (1.13 μg/ml for PCNA, 10 μg/ml for TGF-α, and 4 μg/ml for EGFR) for 1 h at room temperature or 4°C overnight. Biotinylated secondary antibody was applied followed by a streptavidin-horseradish peroxidase conjugate. The immune complexes were visualized with aminoethyl carbazole (AEC) substrate-chromagen. Approximately 20 consecutive sections of tissue were cut for each PT gland. Immunohistochemical staining of PCNA, Ki67, TGF-α, and EGFR proteins was quantified using a Nikon Diaphot-TMD microscope coupled to a camera and an image-analysis system. Images of stained tissue sections were acquired using a DAGE-330 color camera and captured with a Pentium P-166 IBM-compatible computer. The digitized images were converted to a gray scale and analyzed using Image-Pro plus software (Media Cybernetics) according to Mize's study as described before (9, 11). To eliminate variation, the microscope light source intensity used during image capture was kept constant for all sections stained on a given day. The average optical density per section of tissue was calculated by dividing the sum integrated optical density by the sum area.

Statistics

ANOVA was employed to assess statistical differences between all experimental groups under study. Multiple comparisons using the stringent Bonferroni test (or unpaired t-test analysis when indicated) measured the statistical significance of the differences between two experimental groups.

RESULTS

Most of the parathyroid gland enlargement induced by high P in the rat model of kidney disease occurs within the first week after 5/6 nephrectomy (10). Because in normal and transformed human tissues (1, 26), enhanced coexpression of TGF-α and EGFR in a cell type associates with more aggressive growth, initial studies examined the association between increases in EGFR expression and proliferating activity in glands from a prior study (9) in normal and 5/6 nephrectomized rats subjected to dietary P manipulations. Table 1 shows the results of the immunohistochemical quantitation of parathyroid EGFR. Parathyroid EGFR content increased twofold in response to high P within the first week of 5/6 nephrectomy compared with EGFR levels in P-restricted animals, which did not differ significantly from parathyroid EGFR content in rats with normal renal function (sham-operated controls) fed the same high-P diet. The increases in parathyroid EGFR content paralleled those reported for TGF-α. Clearly, kidney disease and high dietary P are required for the rapid (within 1 wk) and marked increases in parathyroid expression of both ligand and receptor of the potent TGF-α/EGFR growth loop.

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Table 1.

Parathyroid EGFR expression in response to changes in dietary P intake

Next, similar studies were conducted in 5/6 nephrectomized rats fed a low-Ca diet (0.1% Ca), a maneuver known to drastically increase parathyroid gland size and serum PTH levels, to address whether simultaneous increases in parathyroid TGF-α and EGFR levels mediated the parathyroid hyperplasia induced by low dietary Ca in early kidney disease. Table 2 shows that the degree of renal failure, as measured by serum creatinine, and serum chemistries were similar in the uremic rats fed low- and high-Ca diets, except for the expected difference in ionized calcium. The rats fed the high-Ca diet served as a negative control for parathyroid growth because high Ca intake completely counteracts the mitogenic signals triggered by the onset of kidney disease. Figure 1 shows a representative immunohistochemical staining for parathyroid PCNA, TGF-α, and EGFR in both experimental groups with high and low Ca intake. The results of the quantitation of each of these proteins are depicted in Table 3. In the uremic rats fed low Ca, the 3.7-fold increase in the number of nuclei staining positive for PCNA was paralleled by a simultaneous 3.2-fold increase in TGF-α and a 2.7-fold increase in EGFR content compared with that in rats with the same degree of renal failure fed a high-Ca diet. Taken together, these results demonstrate a temporal association between simultaneous increases in parathyroid TGF-α and EGFR expression and the high proliferating activity induced by either low dietary Ca or high P intake within the first week of the onset of kidney disease in rats.

Fig. 1.

Parathyroid levels of proliferating cell nuclear antigen (PCNA), transforming growth factor (TGF)-α, and epidermal growth factor receptor (EGFR) expression in response to changes in dietary Ca intake. Representative photomicrographs are shown of immunohistochemical staining of PCNA, TGF-α, and EGFR in parathyroid tissue from 5/6 nephrectomized rats fed either high or low dietary Ca (as described in protocol 1) at day 7 after the onset of renal failure: uremic (U)+high-Ca (U-HCa) diet+V (U-HCa+V); U+low-Ca (U-LCa) diet+vehicle (V; U-LCa+V). Magnification: ×100.

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Table 2.

Serum chemistry and PTH levels

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Table 3.

Parathyroid gland size, PCNA, TGF-α and EGFR expression

To measure the contribution of high P- or low Ca-induced enhancement of parathyroid expression of the TGF-α/EGFR-growth loop to the severity of the hyperplasia in early kidney disease, we utilized the EGFR tyrosine kinase inhibitors AG-1478 and Erlotinib, potent and highly specific inhibitors of growth signals from ligand-activated EGFR (6, 19). In the 5/6 nephrectomized rats fed high dietary P, the P intake was increased from 0.9 to 1.2%/g of diet to aggravate the degree of parathyroid hyperplasia. Initial experiments were conducted using intermittent doses of AG-1478. Table 4 shows serum chemistries from one of the two independent experiments conducted in 5/6 nephrectomized rats (8 and 10/experimental group, respectively) fed 1.2% P/g of diet (high-P diet) and receiving either vehicle (DMSO:PBS; 1:1) or AG-1478 (25 mg/kg body wt) every other day for 1 wk. The dose of AG-1478 had no adverse effect in either study, as judged by no differences in body weight [AG-1478: 161.3 g ± 5.2 vs vehicle: 171.4 ± 0.2 g], serum creatinine, or pH. Ionized Ca and P levels were similar between the uremic rats fed high P receiving vehicle and those treated with AG-1478. The P-restricted group (0.2% P) of 5/6 nephrectomized rats served as a negative control of uremia-induced parathyroid hyperplasia.

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Table 4.

Serum chemistry and PTH levels (protocol IIA)

Table 5 presents the average values from all parathyroid glands harvested. In vivo suppression of growth signals from ligand-activated EGFR, through intermittent (every other day) administration of AG-1478, was sufficient to reduce the proliferating activity (number of PCNA-positive nuclei per gland area) observed in the control group of uremic rats fed high dietary P receiving vehicle by 62%. As a consequence of reduced parathyroid cell growth, serum PTH levels decreased by 43.3% with intermittent AG-1478 administration (Table 4) despite the high dietary P intake. This reduction, however, did not reach statistical significance.

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Table 5.

Parathyroid gland weight and proliferating activity (protocol IIB)

To directly assess whether the incomplete inhibition of high P-induced parathyroid hyperplasia with intermittent (every other day) AG-1478 administration could result, in part, from the reversible binding of this tyrphostin to the EGFR, similar studies were conducted using daily dosing of the small-molecule EGFR tyrosine kinase inhibitor Erlotinib, in phase III clinical trials. The switch from AG-1478 to the quinazolinamine Erlotinib for daily dosing was based in the observed tendency for serum P levels to increase with intermittent AG-1478 administration, despite the reduction in serum PTH. Further elevations in serum P with daily AG-1478 administration would invalidate the experimental approach. Preliminary testing of Erlotinib in the rat model of kidney disease demonstrated no effect of daily dosing of Erlotinib on serum Ca or P levels. Figure 2 (top) shows that high P-induced growth signals were inhibited by daily administration of Erlotinib in a dose-dependent manner. Total inhibition of high P-induced parathyroid growth was achieved with the higher (6 mg/kg body wt) dose of Erlotinib, an antiproliferative potency comparable to that of P restriction when kidney disease was the only stimulus inducing parathyroid cell proliferation. Part of the ability of Erlotinib in suppressing high P-induced growth could result from Erlotinib's inhibition of TGFα self-upregulation, a mechanism associated with aggressive growth, as reported in TGF-α/EGFR-driven carcinomas (3). Figure 2 (bottom) shows that the daily 6 mg/kg ip dose of Erlotinib also prevented the TGF-α-self upregulation induced by kidney disease and high dietary P with an efficacy comparable to that of P restriction in rats in which kidney disease was the only stimulus to enhance parathyroid TGF-α expression and, therefore, cellular growth. Daily doses of up to 6 mg/kg body wt Erlotinib for 1 wk appear to have no adverse effects on 5/6 nephrectomized rats fed a high-P diet, as suggested by the absence of changes in body weight or serum creatinine levels (see Table 6). Values in Table 6 are one of the two sets of similar results from the two independent experiments conducted with Erlotinib and high dietary P.

Fig. 2.

Parathyroid levels of PCNA and TGF-α in response to changes in dietary P intake and anti-EGFR therapy. Immunohistochemical quantitation of PCNA (top) and TGF-α (bottom) in parathyroid tissue from 5/6 nephrectomized rats fed either high (U-HP; n = 11) or low (U-LP; n = 6) dietary P and treated with 3 (U-HP+E3; n = 12) or 6 (U-HP+E6; n = 11) mg Erlotinib/kg body wt, intraperitoneally, daily within the first week of the onset of kidney disease. *P < 0.02 vs. U-HP.

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Table 6.

Body weight and serum chemistries with Erlotinib+dietary P manipulations

The next studies addressed whether the daily ip dose of Erlotinib of 6 mg/kg body wt, capable of preventing high P-induced hyperplasia and TGF-α self-induction, was equally effective in 5/6 nephrectomized rats fed a low-Ca diet. Table 7 shows that low Ca-induced TGF-α expression, self- upregulation, and downstream growth signals (Ki67 expression) in early kidney disease were totally counteracted by prophylactic daily doses of 6 mg/kg body wt of Erlotinib. Table 8 shows that Erlotinib administration had no adverse effects, as suggested by no differences in body weight and serum chemistries between the 5/6 nephrectomized rats fed the low-Ca diet receiving vehicle or 6 mg/kg body wt of Erlotinib.

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Table 7.

Parathyroid gland size and markers of proliferation with Erlotinib+low dietary Ca

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Table 8.

Body weight and serum chemistries with Erlotinib + low dietary Ca

Taken together, these findings demonstrate that growth signals from enhanced parathyroid TGF-α and EGFR expression are indeed a major pathogenic mechanism for the rapid (1 wk) and severe (doubling of gland size) parathyroid hyperplasia secondary to the onset of experimental kidney disease in rats.

DISCUSSION

The present studies in the rat model of kidney disease identified growth signals from enhanced parathyroid expression of TGF-α and EGFR as a major pathogenic mechanism for the hyperplastic growth induced early after the onset of kidney disease and aggravated by high dietary P or low Ca intake. Indeed, increases in parathyroid expression of both components of the powerful TGF-α/EGFR growth loop temporally associate with the enhanced mitogenic activity triggered by kidney disease and worsen by high P or low Ca intake within the first week after 5/6 nephrectomy. More importantly, selective inhibition of TGF-α/EGFR-driven growth in these rats, using the highly specific inhibitors of EGFR tyrosine kinase AG-1478 (2) and CP-358, 774 (19), effectively suppressed parathyroid hyperplasia in a dose-dependent manner. In 5/6 nephrectomized rats fed a high-P diet, prophylactic administration of AG-1478, every other day for 1 wk, reduced parathyroid hyperplasia by 62%. The incomplete suppression of parathyroid cell growth by intermittent administration of AG-1478 suggested either an insufficient anti-EGFR potency of this dose of the tyrphostin or the existence of EGFR-independent growth signals. To directly address the latter possibility, daily dosing of Erlotinib (20) substituted the every other day administration schedule. This regimen should reduce the loss of anti-EGFR potency caused by the reversible binding of the tyrphostin to the EGFR. In fact, daily administration of Erlotinib resulted in a dose-dependent arrest of high-P-induced parathyroid cell growth. The dose of 6 mg Erlotinib/kg body wt completely counteracted the potent mitogenic signals triggered by kidney disease and high dietary P. Furthermore, daily doses of Erlotinib were equally effective in preventing the parathyroid hyperplasia induced by low dietary Ca in early kidney disease. The efficacy of the dose of 6 mg Erlotinib/kg body wt in suppressing high P- and low Ca-induced parathyroid hyperplasia in early kidney disease in rats was comparable to that elicited by either high dietary Ca or P restriction, when kidney disease was the only mitogenic stimuli. This finding conclusively demonstrates a major contribution of enhanced parathyroid expression of TGF-α /EGFR expression and signaling to parathyroid hyperplasia because both dietary maneuvers are known as the most powerful suppressors of the parathyroid hyperplasia induced by kidney disease.

Taken together, these findings demonstrate that growth signals from enhanced parathyroid TGF-α and EGFR content are major determinants of the severe parathyroid hyperplasia induced by kidney disease and aggravated by either high dietary P or low Ca. Two types of growth signaling, cell membrane and novel “nuclear EGFR” signals, are triggered on TGF-α binding to and activation of the EGFR. Cell membrane signals include the Ras/mitogen-activated protein kinase (MAPK) cascade, which causes increased-cyclin D1 activity (28). To initiate nuclear signals, TGF-α-activated EGFR translocates to the nucleus (16), where it acts as a transcription factor (coactivator) to induce cyclinD1 gene transcription. Nuclear EGFR localization associates with severe hyperplastic growth (16). Both membrane and nuclear signals from enhanced TGF-α activation of the EGFR could be operating in patients with secondary hyperparathyroidism because the severity of parathyroid hyperplasia correlates directly with increases in cyclinD1 expression (27). Consequently, downregulation of TGF-α/EGFR expression and cell membrane and nuclear growth signals mediates the antiproliferative properties of high dietary Ca or P restriction. In fact, prevention of the increases in parathyroid levels not only of TGF-α but also of the EGFR accompanied the efficacy of high dietary Ca and P restriction in ameliorating the increases in mitotic activity, parathyroid gland enlargement, and serum PTH levels induced by the onset of kidney disease. This reduction in growth signals from the TGF-α/EGFR loop by high dietary Ca or P restriction in hyperplastic parathyroid cells is a very efficient one. Both maneuvers simultaneously downregulate the expression of the growth promoter TGF-α and its signal transducer, the EGFR. Exclusive downregulation of TGF-α using antisense technology, while capable of reducing hyperproliferative activity in head and neck squamous carcinoma, was ineffective to arrest the growth of normal cells (15). Unfortunately, further characterization of the molecular mechanisms underlying the potent modulation of parathyroid expression of TGF-α and EGFR by dietary manipulations of Ca and P is precluded at present by the lack of a parathyroid cell model. Human and bovine parathyroid cells in culture fail to mimic the in vivo regulation of cellular growth in response to changes in Ca and P concentrations in the incubation media. In fact, primary cultures of hyperplasic human parathyroid cells increase rather than suppress growth with elevations in calcium concentration in the incubation media (21).

In tumors in which growth is driven by TGF-α EGFR overexpression, TGF-α activation of the EGFR also results in further stimulation of TGF-α gene transcription (3, 7), thus generating a positive feedback loop that furthers cell growth. Anti-EGFR therapy allowed us to examine whether TGF-α activation of the EGFR was involved in the control of TGF-α expression in the parathyroid glands. Inhibition of TGF-α/EGFR-signaling through daily doses of Erlotinib to 5/6 nephrectomized rats fed either a high-P or a low-Ca diet prevented TGF-α/EGFR growth signals and also TGF-α induction of its own expression in the parathyroid glands. Thus TGF-α self-stimulation also occurs in the parathyroid hyperplasia secondary to kidney disease and is aggravated by both high dietary P and low Ca intake.

In summary, the enhancement of parathyroid TGF-α and EGFR expression and growth signals occurs early after the onset of kidney disease in rats, is aggravated by high-P or low-Ca diets, and constitutes the major cause of TGF-α self-induction and severe parathyroid hyperplasia.

GRANTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-062713 (to A. S. Dusso) and grants from Research in Renal Diseases, Washington University, and Abbott Pharmaceuticals (to E. Slatopolsky). T. Sato is a recipient of an award by the Nagono Medical Foundation, Nagoya, Japan. I. Gonzalez Suarez is a recipient of a scholarship for Biomedical Research to Hospital Central de Asturias from the Ministerio de Sanidad y Consumo, Spain.

Acknowledgments

The authors thank Jane Boudreaux and Daniel Martin for assistance in 5/6 nephrectomies and Julie Hilker for help with animal care and sample processing.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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