How one TSH receptor antibody induces thyrocyte proliferation while another induces apoptosis

Abstract

Thyroid stimulating hormone (TSH) activates two major G-protein arms, Gsα and Gq leading to initiation of down-stream signaling cascades for survival, proliferation and production of thyroid hormones. Antibodies to the TSH receptor (TSHR-Abs), found in patients with Graves' disease, may have stimulating, blocking, or neutral actions on the thyroid cell. We have shown previously that such TSHR-Abs are distinct signaling imprints after binding to the TSHR and that such events can have variable functional consequences for the cell. In particular, there is a great contrast between stimulating (S) TSHR-Abs, which induce thyroid hormone synthesis and secretion as well as thyroid cell proliferation, compared to so called "neutral" (N) TSHR-Abs which may induce thyroid cell apoptosis via reactive oxygen species (ROS) generation. In the present study, using a rat thyrocyte (FRTL-5) ex vivo model system, our hypothesis was that while N-TSHR-Abs can induce apoptosis via activation of mitochondrial ROS (mROS), the S-TSHR-Abs are able to stimulate cell survival and avoid apoptosis by actively suppressing mROS. Using fluorescent microscopy, fluorometry, live cell imaging, immunohistochemistry and immunoblot assays, we have observed that S-TSHR-Abs do indeed suppress mROS and cellular stress and this suppression is exerted via activation of the PKA/CREB and AKT/mTOR/S6K signaling cascades. Activation of these signaling cascades, with the suppression of mROS, initiated cell proliferation. In sharp contrast, a failure to activate these signaling cascades with increased activation of mROS induced by N-TSHR-Abs resulted in thyroid cell apoptosis. Our current findings indicated that signaling diversity induced by different TSHR-Abs regulated thyroid cell fate. While S-TSHR-Abs may rescue cells from apoptosis and induce thyrocyte proliferation, N-TSHR-Abs aggravate the local inflammatory infiltrate within the thyroid gland, or in the retro-orbit, by inducing cellular apoptosis; a phenomenon known to activate innate and by-stander immune-reactivity via DNA release from the apoptotic cells.

Keywords: Antibodies; Apoptosis; Proliferation; ROS-signaling; TSH receptor; Thyrocyte.

Fig. 1

Immunohistochemistry and live imaging of stress markers in thyrocytes. Panel A: Immunohistochemical detection of stress-induced proteins and ROS. Rabbit polyclonal primary antibody was used to detect Mn-SOD in N-TSHR-mAb (IC8 1ug/ml) treated (24 h) FRTL-5 thyrocytes. The N-TSHR-mAb also induced heat shock proteins (HSP) 70 and 60 as detected by mAbs against each protein. HSP70 and 60 are normally localized within the endoplasmic reticulum and mitochondria, respectively. Such proteins were not induced by an isotype control mAb (see insets). Scale bar on images corresponds to 20micron. Panel B: Live imaging of ROS in thyrocytes using three independent dyes. Cells were treated for 24 h with N-TSHR-mAb (IC8 1ug/ml). Representative images of both D123 and H₂DCFDA showed diffuse staining throughout the cell cytoplasm. Some perinuclear condensations were also documented. The distribution of staining patterns paralleled mitochondrial ROS (mROS). Nucleoli are indicated as N.

Fig. 2

Apoptosis induced by N-mAb but not by St-mAb or TSH. Panels A & B: Both total caspases and Annexin V assays indicated that N-mAb (IC8) was capable of inducing apoptosis via ROS induction. Dose-dependent induction of total caspases was confirmed by FLICA assay (Panel A). Staurosporine (STP) was used as a positive control for apoptosis. Neutral antibody induced total caspase activity was significantly enhanced compared to control antibody treated cells. Live-imaging analyses (Panel B) indicated activation of both mROS (red) and Annexin V (green). Representative images indicated co-localization of mROS (red) and Annexin V (green) by yellow color (connected arrow). Nuclear fragmentation was also observed in some apoptotic cells (arrow). Panels C & D: Suppression of mROS induced by S-TSHR-mAb (M22 1ug/ml) or TSH (1mU/ml) overtime. The peak level of suppression was noted on day 3 while N-TSHR-mAb (IC8) continued to activate mROS (Panel C). S-TSHR-mAb did not induce any apoptosis as there was no binding of Annexin V by live-imaging (Panel D) (lack of green) but cell proliferation was induced on day 3 as indicated in our previous study [2]. A few cells showed a minor induction of mROS (red; arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

S-TSHR-mAb suppressed mROS production while N-TSHR-mAb induced mROS. Panel A: Live fluorometric analyses indicated that S-TSHR-mAb (M22) suppressed mROS (red) in a dose-dependent manner while N-TSHR-mAb (IC8) had the opposite effect (insets). Nucleoli were stained with live nuclear dye (Hoechst 33342). Panel B: Cyclic AMP (32 pmol/ml), IBMX (1 mM), Forskolin (10 μM), and TSH (1 mU/ml) suppressed significantly mROS induction by N-TSHR-mAb. Panel C: In contrast, PKA inhibitor (H89) induced mROS expression in a time dependent manner followed by apoptosis (not shown). Panel D: The S-TSHR-mAb (1ug/ml) suppressed mROS induction at different concentrations of PKA inhibitor. Panel E: The S-TSHR-mAb was also capable of rescuing cells from PKA inhibitor-induced thyrocyte apoptosis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Multiple phosphoproteins were detected by Immunoblot and immunohistochemistry. Panel A – Representative immunoblot showing activation of multiple signaling proteins. Activation of different signaling molecules was consistent with our earlier immunoblot studies (1,2). Panel B: Activated signals were inconsistent with time. The N-TSHR-mAb failed to maintain sustained activation of some important key signaling molecules while the S-TSHR-mAb and TSH demonstrated sustained activation of their signaling cascades. Importantly, the S-TSHR-mAb induced a sustained activation of AKT/mTOR/S6/ERK_1/2 and PKA/CREB signaling molecules at 24 h. By contrast, the N-TSHR-mAb continued to activate a stress related signaling molecule p38 and failed to show sustained activities of AKT/mTOR/S6/ERK_1/2 and PKA/CREB. C = Control mAb, S = S-TSHR-mAb (M22), TSH = thyroid stimulating hormone, N = N-TSHR-mAb (IC8). Panel C: Immunohistochemistry of phospho-CREB protein (Green) using an anti-phosphorylated CREB antibody revealed nuclear accumulation induced by S-TSHR-mAb not seen in N-mAb treated cells. Nucleus was stained with blue dye. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Representative views of ROS and apoptosis induced by human immunoglobulins (IgGs) from patients with Grave’ disease. Rat thyroid cells were treated twice with purified human IgG (10 μg/ml) on day 3 and day 5 of culture. They were examined daily and imaged on day 7 when loss of cells with apoptotic features appeared to begin. Induction of ROS was prominent in three patients (GD-1, -3 & -4) compared to two other patients (GD-2 &-5). Two patients (GD-2 & GD-4) showed loss of cells with apoptotic features of nuclear fragmentation while showing lower ROS staining (GD-2) while the other (GD-4) had loss of nuclear dye (Hoecst 33342) with a higher content of diffuse cytoplasmic ROS staining. IgG from patient GD-5 did not produce such effects and showed less ROS compared to even control IgG treated cells. Control = normal human IgG, GD1-5 are IgG preparations from 5 patients with Graves’ disease and positive TSR-Ab assay.

Fig. 6

A cycle of Graves’ autoimmunity generated by two mutually non-exclusive mechanisms exacerbated by multiple TSHR-Abs. S-TSHR-Abs induce a living (proliferation) cycle while N-TSHR-Abs involve a death (apoptosis) cycle. The living cycle includes activation of PKA/CREB/AKT leading to cell proliferation and excessive T3/T4 production while suppressing mitoROS and NADPH oxidase. In contrast, the death cycle has the opposite effects. Neutral antibodies suppress PKA/CREB/AKT leading to activation of mitoROS and induction of apoptosis and cell death.