Altered ion transport by thyroid epithelia from CFTR(-/-) pigs suggests mechanisms for hypothyroidism in cystic fibrosis

Abstract

Subclinical hypothyroidism has been linked to cystic fibrosis, and the cystic fibrosis transmembrane conductance regulator (CFTR) shown to be expressed in the thyroid. The thyroid epithelium secretes Cl⁻ and absorbs Na(+) in response to cAMP. Chloride secretion may provide a counter-ion for the SLC26A4 (pendrin)-mediated I⁻ secretion which is required for the first step of thyroid hormonogenesis, thyroglobulin iodination. In contrast, few models exist to explain a role for Na(+) absorption. Whether CFTR mediates the secretory Cl⁻ current in thyroid epithelium has not been directly addressed. We used thyroids from a novel pig CFTR(-/-) model, generated primary pig thyroid epithelial cell cultures (pThECs), analysed these cultures for preservation of thyroid-specific transcripts and proteins, and monitored the following parameters: (1) the Cl⁻ secretory response to the cAMP agonist, isoprenaline; and (2) the amiloride-sensitive Na(+) current. Baseline short-circuit current (I(sc)) did not differ between CFTR(+/+) and CFTR(-/-) cultures. Serosal isoprenaline increased I(sc) in CFTR(+/+), but not CFTR(-/-), monolayers. Compared with CFTR(+/+) thyroid cultures, amiloride-sensitive Na(+) absorption measured in CFTR(-/-) pThECs represented a greater fraction of the resting I(sc). However, levels of transcripts encoding epithelial sodium channel (ENaC) subunits did not differ between CFTR(+/+) and CFTR(-/-) pThECs. Immunoblot analysis verified ENaC subunit protein expression, but quantification indicated no difference in expression levels. Our studies definitively demonstrate that CFTR mediates cAMP-stimulated Cl⁻ secretion in a well-differentiated thyroid culture model and that knockout of CFTR promotes increased Na(+) absorption by a mechanism other than increased ENaC expression. These findings suggest several models for the mechanism of cystic fibrosis-associated hypothyroidism.

Figure 1

A) Histology and B) growth of litter-matched, 1-day-old CFTR ^+/+ and CFTR ^−/− pig thyroids. Both image sets were taken at the same magnification; scale bar, 100 µm. A) HE staining of paraffin sections prepared from litter-matched, neonatal pig thyroids. B) Phase image of pThEC cultures after 1 week of growth.

Figure 2

Expression of thyroid-specific proteins by CFTR ^+/+ and CFTR ^−/− pThECs. Cultures were prepared from three separate litter-matched pairs. The normalized expression levels of the two forms of NIS are shown in panels A) and B); neither form was expressed differently (p = 0.32 for the 100 kDa form and 0.19 for the 73 kDa species. Panel C) ClC-5 expression likewise did not differ between the CFTR ^+/+ and CFTR ^−/− pThECs (p = 0.40). Panels E), F) and G) summarize relative levels of thyroglobulin (Tg), DUOX1 and thyroperoxidase (TPO), all which did not reveal significant differences (p = 0.09, 0.82 and 0.47, respectively). H) Expression of the Na⁺/K⁺ ATPase was obtained as an approximate index of transport capacity; no difference between the two groups was found. All measurements were obtained by normalization to the respective actin signal (D). All p values were obtained using paired t-tests.

Figure 3

I_sc recordings show the presence of isoproterenol-activated I_sc in A) CFTR ^+/+, but not B) CFTR ^−/− pThECs. Addition of isoproterenol is indicated by arrow labeled “iso”. Note that the baseline I_sc is inhibited swiftly by addition of mucosal amiloride (10⁻⁵ M; “amil”) to both wild type and knockout monolayers. The traces are responses of monolayers grown from 1-day-old littermates and studied at day 14 post-plating. Baseline I_sc values were 3.45 µA.cm² and 4.705 µA.cm² for this pair of CFTR ^+/+ and CFTR ^−/− pThEC monolayers, respectively. The amiloride-sensitive fraction of I_sc for the CFTR ^+/+ pThEC monolayer depicted here was 0.63. For the CFTR ^−/− trace shown, this was 0.74. C) Summarized peak responses to serosal isoproterenol (10⁻⁵ M) are shown for CFTR ^+/+ pThEC monolayers (left; n = 9 monolayers from 5 preparations). For comparison, data from CFTR ^−/− pThECs are provided (right; n = 5 monolayers from 3 preparations). D) Fractional amiloride-sensitive I_sc is significantly greater in pThECs grown from CFTR ^−/− thyroids (0.79 ± 0.007; right) compared to that measured in CFTR ^+/+ thyroid-derived monolayers (0.55 ± 0.048; left). Data represent the mean values ± SEM.

Figure 4

Quantitative immunoblot analysis shows that CFTR ^+/+ and CFTR ^−/− pThECs express ENaC subunit proteins at similar levels. A) Relative levels of the α-ENaC 95 kDa and B) 32 kDa species were not altered in CFTR ^−/− cells compared CFTR ^+/+ (p = 0.44 and 0.30, respectively). This was similarly the case for C) β-ENaC (p = 0.28) and D) γ-ENaC (83 kDa) (p = 0.70). Panel E) depicts the corresponding control probes for CFTR and actin. All p values were obtained using paired t-tests on n = 3 litter-matched pairs.

Figure 5

Hypothetical models linking CFTR function to thyroid hormonogenesis are shown in this schematic diagram. Panel A shows hypothesized CFTR functions. Leftmost diagram depicts the situation in which CFTR directly conducts I⁻. Middle diagram shows CFTR regulates SLC26A4/Pendrin-mediated I⁻ movement, possibly by STAS domain interactions and transport of counter-anions for SLC26A4. Right diagram shows the situation whereby CFTR regulates Na⁺ entry through ENaC, thereby preserving the Na⁺ gradient that is required for NIS-mediated I⁻ uptake. Panel B illustrates predicted outcomes of CFTR ablation, based on each of the schemes illustrated in Panel A.