Understanding Nis Regulation In Health And Disease May Improve The Effectiveness Of Radioiodide Treatment

The Natural Thyroid Diet

The Natural Thyroid Diet

Get Instant Access

TSH and are the two main factors that regulate NIS expression: TSH stimulates and I" decreases it. Hence, TSH stimulation and I" depletion ofresidual thyroid carcinoma tissue are the two most important modulators routinely used to optimize radioiodide treatment. To achieve maximum therapeutic effect, thyroidectomized patients must have TSH levels above 30 mU/l and must have been on a low I" diet for two weeks prior to initiation of radioiodide treatment (1).

TSH has long been known to be a key regulator not only of NIS expression but also of thyroidal I" uptake (i.e., NIS activity). No thyroidal NIS expression is observed in hypophysectomized rats (because of the lack of TSH), but thyroidal NIS expression is restored as early as 24 h after treatment with TSH. In intact (i.e., non-hypophysectomized) rats, treatment with the organification inhibitor propylth-iouracil causes elevated TSH levels, which in turn lead to higher NIS expression than in control animals (10). TSH regulates NIS expression at both the transcriptional and posttranscriptional levels. Several groups have demonstrated that TSH upregulates I" transport by a cAMP-mediated increase in NIS transcription, while withdrawing TSH causes decreased cAMP levels and diminished NIS transcription (33).

Figure 2. A: NIS immunohistochemistry in Graves' disease. NIS is localized in the basolateral plasmamembrane B: NIS immunohistochemistry in follicular carcinoma shows the intracellular localization of the significantly overexpressed NIS protein C: Indirect immunofluorescence analysis of NIS localized in the plasma membrane of FRTL-5 cells kept in the presence of TSH, D: intracellular NIS localization in TSH deprived FRTL-5 cells E: schematic representation of NIS plasma membrane localization and iodide transport in FRTL-5 cells kept in the presence of TSH F: schematic representation of NIS localized in the intracellular membrane compartments in TSH deprived FRTL-5 cells, resulting in lack of iodide transport.

Figure 2. A: NIS immunohistochemistry in Graves' disease. NIS is localized in the basolateral plasmamembrane B: NIS immunohistochemistry in follicular carcinoma shows the intracellular localization of the significantly overexpressed NIS protein C: Indirect immunofluorescence analysis of NIS localized in the plasma membrane of FRTL-5 cells kept in the presence of TSH, D: intracellular NIS localization in TSH deprived FRTL-5 cells E: schematic representation of NIS plasma membrane localization and iodide transport in FRTL-5 cells kept in the presence of TSH F: schematic representation of NIS localized in the intracellular membrane compartments in TSH deprived FRTL-5 cells, resulting in lack of iodide transport.

The detailed analysis of the rat and human NIS promoters has confirmed the significant role of Pax8 in NIS expression (34-36). In rat, the proximal NIS promoter was found to contain a TTF1 binding site and a TSH-responsive element where a putative transcription factor NTF-1 (NIS TSH-responsive factor 1) interacts (39). NIS upstream enhancer (NUE-2495 to -2260) contains two Pax-8 binding sites and a degenerate CRE (cAMP-responsive element sequence), which are essential for full TSH cAMP-dependent transcription of NIS (34). Interestingly, during chronic TSH stimulation when the catalytic subunit of PKA is downregulated, cAMP is still able to stimulate NIS transcription, indicating the existence of both PKA-dependent and independent mechanisms (34). Recently, a thyroid-specific far-upstream (-9847 to -8968) enhancer in the human NIS gene - highly homologous to the rat NUE - has been reported. It contains putative Pax-8 and TTF-1 binding sites and a CRE-like sequence. The TTF-1 binding site is not required for full activity (35, 36).

NIS gene

| transcription

NI5 mRNA

I translation

NIS protein biosynthesis

^ posttranslational modifications NIS protein maturation i plasma membrane targeting

Functional NI5 in the p I as ma membrane retention in the plasma membrane

NIS retrieved from the plasma mem brans

| protein degradation degradation Figure 3. Multiple levels of NIS regulation.

FRTL-5 cells are rat-thyroid-derived, well-differentiated normal thyroid epithelial cells that grow in media supplemented with TSH. These cells are frequently used as an in vitro model system to study TSH regulation. In FRTL-5 cells, NIS expression is TSH dependent. Kaminsky et al. (37) observed that, in the absence of TSH in the medium, intact FRTL-5 cells did not transport I", whereas membrane vesicles prepared from the same TSH-deprived cells surprisingly maintained their I" transporting ability. This suggested that mechanisms other than transcriptional might also operate in regulating NIS activity in response to TSH. Riedel et al. (22) investigated this phenomenon in detail. They observed that in the absence of TSH, there was no de novo NIS synthesis in FRTL-5 cells, while previously synthesized NIS was redistributed from the plasma membrane to intracellular membrane compartments. (Figure 2 C,D,E,F) These authors also demonstrated that NIS has a long half-life: 5 days in the presence and 3 days in the absence of TSH. Considering the TSH regulation of NIS expression and the long halflife of NIS, it is clear that NIS mRNA levels alone are not a good indicator of actual NIS protein levels. Instead, NIS protein levels must be assessed directly with anti-NIS Abs. In addition, it is also essential to keep in mind that NIS protein expression, in turn, does not necessarily correlate with NIS activity, because such factors as subcellular distribution of NIS to the plasma membrane play a key role in NIS function; hence, it is crucial to quantitate NIS activity (Figure 3).

TSH modulates NIS phosphorylation

The mechanism by which TSH regulates the subcellular distribution of NIS is unknown. Phosphorylation has been shown to be implicated in the activation and subcellular distribution of several transporters (38-40). NIS has several consensus sites for kinases, including those for cAMP-dependent protein kinase, protein kinase C, and casein kinase-2 (9, 22). Furthermore, TSH actions in the thyroid are mainly mediated by cAMP. All these points raised the possibility that phosphorylation might be involved in the regulation of NIS subcellular distribution. When FRTL-5 cells were labeled with lysed, and immunoprecipitated with anti-NIS Ab, it was observed in the autoradiogram that NIS was phosphorylated, independently of the presence of TSH in the culture medium (22). The phosphopeptide map obtained after NIS digestion with trypsin was markedly different when TSH was present from that when TSH was absent (22).

The predominant phosphorylated region of NIS was determined by treatment of the immunoprecipitated symporter with CNBr. CNBr cleaves polypeptides at methionine residues. The anti-NIS Ab generated against the last 16 amino acids ofNIS recognized an 11-kDa polypeptide observed also by autoradiography. The densitometric quantitation of the autoradiogram indicated that the major phosphorylation region of NIS is the carboxy terminus (22). Moreover, TSH increased the phosphorylation level of the COOH terminus of NIS ~ 16-fold. For the identification of which of the serine residues within the COOH terminus are phosphorylated, S551, S552, S568, and S581 (Fig. 1) were replaced individually and simultaneously with alanine. Significantly, the replacement of the four serines of the COOH terminus promoted phosphorylation of threonines in NIS, suggesting that there is an important biological pressure to preserve phosphorylation of NIS at the COOH terminus (22). Future experiments should elucidate whether NIS phosphorylation is involved in trafficking and/or retention of NIS at the plasma membrane.

Regulation of NIS expression by I

As indicated above, I" itself, the substrate of NIS, also regulates NIS expression, but the mechanism of this regulation is less clear than that of TSH. For over 60 years, it has been known that organification and, consequently, thyroid hormone biosynthesis, are blocked when the intracellular concentration of I" rises to a certain threshold. This phenomenon (i.e., the inhibition of I" organification by a high concentration of I") is called the Wolff-Chaikoff effect, and it has been used to block thyroid function in hyperthyroid patients (41, 42). I also suppresses I transport in a time- and dose-dependent manner, an effect that has been investigated by several groups both in vitro and in vivo (43-45). As I" transport decreases, the intrathyroidal I" concentration falls, the inhibition of organification is relieved, and thyroid hormone synthesis resumes; thus, by downregulating its own I" transport, the thyroid "escapes" from the inhibitory effect of overload. Grollmann et al (45) investigated the effect of I" preincubation on uptake in FRTL-5 cells, and found that preincubation of these cells suppresses uptake in a dose- and time-dependent manner. These authors observed decreased I" transport after a 2-h incubation with 100 |aM of NaI (45)

With the availability of the NIS cDNA and anti-NIS Abs, the inhibitory effect of on its own transport in the thyroid has been partially reinvestigated by several groups. In dog, Uytterspot et al (46) found that I" inhibited both TPO and NIS

mRNA expression, but no protein levels were measured. In FRTL-5 cells, Spitzweg et al (47) and Eng et al (48) published somewhat contradictory results. Spitzweg et al (47) reported a 50% decrease in I" uptake. However, in these uptake studies, the specific activity of the radioactive used in the transport measurements was diluted out by preincubation with unlabeled which results in its intracellular accumulation. Without taking this factor into account, the interpretation ofthese findings is uncertain. These authors also reported a decrease in NIS mRNA levels, but did not determine NIS protein. In contrast, Eng et al. (48) did not find decreased NIS mRNA levels after preincubation. Instead, they found that both the levels and the half-life of the NIS protein were significantly decreased. Hence, Eng et al. (48) concluded that regulates its own transport in FRTL-5 cells mostly by posttranscriptional mechanisms. Surprisingly, the same authors found that both NIS mRNA and protein levels were decreased in vivo in response to the administration of I" (49). Evidently, a thorough molecular examination of the regulatory effect of on transport simultaneously assessing NIS expression, subcellular localization, and kinetic properties is required to understand the intriguing role of I" in its own transport.

Effect of spatial organization of thyroid cells

It is clear from earlier studies that the spatial organization and apical-basolateral polarization of thyroid epithelial cells significantly influence their functions, including transport, organification, and protein expression. Roger et al (50) isolated human thyroid epithelial cells from normal subjects and grew the cells in the presence or absence of serum. They observed that, whereas cells grown in the presence of serum formed monolayers, in the absence of serum the cells formed aggregates. Following TSH stimulation, cells in aggregates exhibited more avid I" transport than cells in monolayers. Interestingly, this TSH stimulatory effect was abolished by the addition of serum to the medium. Takasu et al (51) showed that, in porcine thyroid cells, polarity is important for uptake, and a follicular structure is required for organification. Kogai et al (52) have recently reinvestigated the effect of spatial organization of thyro-cytes on NIS expression and function. They showed that TSH upregulates both NIS mRNA and protein levels in 2- and 3-dimensional human thyrocyte primary cultures, but a significant increase in uptake occurs only in 3-dimensional structures.

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment