Estradiol differentially regulates calreticulin: a potential link with abnormal T cell function in systemic lupus erythematosus?

Abstract

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease that affects women nine times more often than men. The present study investigates estradiol-dependent control of the calcium-buffering protein, calreticulin, to gain further insight into the molecular basis of abnormal T cell signaling in SLE T cells.

Methods: T cells were purified from blood samples obtained from healthy females and SLE patients. Calreticulin expression was quantified by real-time polymerase chain amplification. Calreticulin and estrogen receptor-α were co-precipitated and analyzed by Western blotting to determine if the proteins associate in T cells.

Results: Calreticulin expression increased (p = 0.034) in activated control T cells, while estradiol decreased (p = 0.044) calreticulin in resting T cells. Calreticulin expression decreased in activated SLE T cell samples and increased in approximately 50% of resting T cell samples. Plasma estradiol was similar (p > 0.05) among SLE patients and control volunteers. Estrogen receptor-α and calreticulin co-precipitated from nuclear and cytoplasmic T cell compartments.

Conclusions: The results indicate that estradiol tightly regulates calreticulin expression in normal human T cells, and the dynamics are different between activated and resting T cells. The absence of this tight regulation in SLE T cells could contribute to abnormal T cell function.

Keywords: SLE; calreticulin; estradiol; estrogen receptor-α; human T cells.

Figure 1

Calreticulin expression increases in response to physiological concentrations of estradiol in activated T cells. T cells were isolated from healthy volunteers and cultured for 24 h without (-E) or with three physiological concentrations (10⁻⁹ to 10⁻⁷ M estradiol). The T cells were activated during the last 4 h of culture. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR from samples in triplicate. Calreticulin increased at the three doses tested with maximum expression at 10⁻⁸ M estradiol. Data are mean values +/− SD. (B) Calreticulin protein expression increased in response to estradiol at the three physiological concentrations tested. The amount of proteins was greatest (1.9-fold increase) at 10⁻⁸ M estradiol stimulation. Data shown are representative of two independent experiments.

Figure 1

Calreticulin expression increases in response to physiological concentrations of estradiol in activated T cells. T cells were isolated from healthy volunteers and cultured for 24 h without (-E) or with three physiological concentrations (10⁻⁹ to 10⁻⁷ M estradiol). The T cells were activated during the last 4 h of culture. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR from samples in triplicate. Calreticulin increased at the three doses tested with maximum expression at 10⁻⁸ M estradiol. Data are mean values +/− SD. (B) Calreticulin protein expression increased in response to estradiol at the three physiological concentrations tested. The amount of proteins was greatest (1.9-fold increase) at 10⁻⁸ M estradiol stimulation. Data shown are representative of two independent experiments.

Figure 2

Calreticulin expression increases temporally in response to estradiol in activated T cells. T cells were isolated from healthy volunteers and cultured in estradiol (10⁻⁷ M) containing medium for the indicated times. The T cells were activated during the last 4 h of culture. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR from samples in triplicate. Data are mean values +/− SD. (B) Calreticulin protein increased in response to estradiol as assessed by Western blotting. The amount of protein increased approximately 1.7-fold at 6 h after stimulation and remained increased (1.8-fold) at 24 h. Data shown are representative of two independent experiments.

Figure 2

Calreticulin expression increases temporally in response to estradiol in activated T cells. T cells were isolated from healthy volunteers and cultured in estradiol (10⁻⁷ M) containing medium for the indicated times. The T cells were activated during the last 4 h of culture. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR from samples in triplicate. Data are mean values +/− SD. (B) Calreticulin protein increased in response to estradiol as assessed by Western blotting. The amount of protein increased approximately 1.7-fold at 6 h after stimulation and remained increased (1.8-fold) at 24 h. Data shown are representative of two independent experiments.

Figure 3

Calreticulin expression increased (p = 0.028) in response to estradiol in activated control T cell samples. T cell samples obtained from normal volunteers were cultured for 18 h without and with estradiol. The samples were activated for 4 h as described in the text and the amount of calreticulin in the same samples without or with estradiol was compared using real time PCR. Data are mean values +/− SD from samples in triplicate. Black bars are data from T cells cultured with estradiol and white bars are data from T cells cultured without estradiol.

Figure 4

Calreticulin expression does not change (p = 0.87) in response to estradiol in activated SLE T cell samples. T cell samples obtained from SLE patients were cultured for 18 h without and with estradiol. The samples were activated for 4 h and the amount of calreticulin in the same samples without or with estradiol was compared using real time PCR. Data are mean values +/− SD from samples in triplicate. Black bars are data from T cells cultured with estradiol and white bars are data from T cells cultured without estradiol.

Figure 5

Calreticulin expression decreases at 24 h in resting T cells. T cells were isolated from healthy volunteers and cultured in medium containing estradiol (10⁻⁷ M) for the indicated times. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR. Data are mean values +/− SD of samples in triplicate. (B) Calreticulin protein increased in response to estradiol at 6 and 12 h post stimulation as assessed by Western blotting. The protein declined at 24h post stimulation to levels similar to that of T cells cultured without estradiol. Data shown are representative of two independent experiments.

Figure 5

Calreticulin expression decreases at 24 h in resting T cells. T cells were isolated from healthy volunteers and cultured in medium containing estradiol (10⁻⁷ M) for the indicated times. RNA and protein was sequentially separated as detailed in the text. (A) Calreticulin mRNA was measured using real-time PCR. Data are mean values +/− SD of samples in triplicate. (B) Calreticulin protein increased in response to estradiol at 6 and 12 h post stimulation as assessed by Western blotting. The protein declined at 24h post stimulation to levels similar to that of T cells cultured without estradiol. Data shown are representative of two independent experiments.

Figure 6

Calreticulin expression is downregulated (p = 0.044) by estradiol in resting control T cells. T cell samples obtained from normal volunteers were cultured for 18 h without and with estradiol. The amount of calreticulin in the same samples without or with estradiol was compared using real time PCR. Data are mean values +/− SD from samples in triplicate. Black bars are data from T cells cultured with estradiol and white bars are data from T cells cultured without estradiol.

Figure 7

Calreticulin expression does not change (p = 0.77) in response to estradiol in resting SLE T cell samples. T cell samples were obtained from SLE patients and cultured without and with estradiol. The amount of calreticulin in the same samples without or with estradiol was compared using real time PCR. Data are mean values +/− SD from samples in triplicate. Black bars are data from T cells cultured with estradiol and white bars are data from T cells cultured without estradiol.

Figure 8

Calreticulin and estrogen receptor-α associate in T47D breast cancer cells and human T cells. (A) Separation of nuclear and cytosolic fractions from T47D breast cancer cells shows enrichment of histone deacetylase (HDAC) in the nuclear fraction. T47D breast cancer cells were homogenized and cytoplasmic and nuclear fractions were separated. The extracts were size fractioned by SDS-PAGE and the proteins were transferred to nitrocellulose by standard methods. The membrane was reacted with an antibody to detect HDAC which migrated with an apparent molecular mass of 62 kDa. Arrows indicate the position of molecular size standards. (B) Calreticulin and estrogen receptor-α associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody which detected a protein at 68 kDa. Lane 1, molecular size standards; Lane 2, no extract, negative control; Lane 3, total T47D extract (10 µg); Lane 4, T47D cytosolic extract; Lane 5, T47D nuclear extract. Data shown are representative of three independent experiments. (C) Estrogen receptor-α and calreticulin associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Estrogen receptor-α was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with a calreticulin antibody which detected a protein at 58 kDα. Lane 1, total T47D extract, positive control (10 µg); Lane 2, T47D cytosolic extract; Lane 3, T47D nuclear extract. Arrows indicated the position of the molecular size standards. Data shown are representative of two independent experiments. (D) Calreticulin and estrogen receptor-α associate in normal resting T cells. T cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody. Lane 1, T47D nuclear extract, positive control, 10 µg); Lane 2, T cell cytosolic extract; Lane 3, T cell nuclear extract. Arrows indicate positions of molecular size standards. The size of the estrogen receptor-α on the immunoblots was approximately 68 kDa. Data shown are representative of two independent T cell samples.

Figure 8

Calreticulin and estrogen receptor-α associate in T47D breast cancer cells and human T cells. (A) Separation of nuclear and cytosolic fractions from T47D breast cancer cells shows enrichment of histone deacetylase (HDAC) in the nuclear fraction. T47D breast cancer cells were homogenized and cytoplasmic and nuclear fractions were separated. The extracts were size fractioned by SDS-PAGE and the proteins were transferred to nitrocellulose by standard methods. The membrane was reacted with an antibody to detect HDAC which migrated with an apparent molecular mass of 62 kDa. Arrows indicate the position of molecular size standards. (B) Calreticulin and estrogen receptor-α associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody which detected a protein at 68 kDa. Lane 1, molecular size standards; Lane 2, no extract, negative control; Lane 3, total T47D extract (10 µg); Lane 4, T47D cytosolic extract; Lane 5, T47D nuclear extract. Data shown are representative of three independent experiments. (C) Estrogen receptor-α and calreticulin associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Estrogen receptor-α was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with a calreticulin antibody which detected a protein at 58 kDα. Lane 1, total T47D extract, positive control (10 µg); Lane 2, T47D cytosolic extract; Lane 3, T47D nuclear extract. Arrows indicated the position of the molecular size standards. Data shown are representative of two independent experiments. (D) Calreticulin and estrogen receptor-α associate in normal resting T cells. T cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody. Lane 1, T47D nuclear extract, positive control, 10 µg); Lane 2, T cell cytosolic extract; Lane 3, T cell nuclear extract. Arrows indicate positions of molecular size standards. The size of the estrogen receptor-α on the immunoblots was approximately 68 kDa. Data shown are representative of two independent T cell samples.

Figure 8

Calreticulin and estrogen receptor-α associate in T47D breast cancer cells and human T cells. (A) Separation of nuclear and cytosolic fractions from T47D breast cancer cells shows enrichment of histone deacetylase (HDAC) in the nuclear fraction. T47D breast cancer cells were homogenized and cytoplasmic and nuclear fractions were separated. The extracts were size fractioned by SDS-PAGE and the proteins were transferred to nitrocellulose by standard methods. The membrane was reacted with an antibody to detect HDAC which migrated with an apparent molecular mass of 62 kDa. Arrows indicate the position of molecular size standards. (B) Calreticulin and estrogen receptor-α associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody which detected a protein at 68 kDa. Lane 1, molecular size standards; Lane 2, no extract, negative control; Lane 3, total T47D extract (10 µg); Lane 4, T47D cytosolic extract; Lane 5, T47D nuclear extract. Data shown are representative of three independent experiments. (C) Estrogen receptor-α and calreticulin associate in T47D breast cancer cells. T47D breast cancer cells were homogenized and nuclear and cytoplasmic fractions were separated. Estrogen receptor-α was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with a calreticulin antibody which detected a protein at 58 kDα. Lane 1, total T47D extract, positive control (10 µg); Lane 2, T47D cytosolic extract; Lane 3, T47D nuclear extract. Arrows indicated the position of the molecular size standards. Data shown are representative of two independent experiments. (D) Calreticulin and estrogen receptor-α associate in normal resting T cells. T cells were homogenized and nuclear and cytoplasmic fractions were separated. Calreticulin was immunoprecipitated and the immunoprecipitates were size fractionated by SDS-PAGE. Proteins in the immunoprecipitates were transferred to nitrocellulose and the membrane was reacted with an estrogen receptor-α antibody. Lane 1, T47D nuclear extract, positive control, 10 µg); Lane 2, T cell cytosolic extract; Lane 3, T cell nuclear extract. Arrows indicate positions of molecular size standards. The size of the estrogen receptor-α on the immunoblots was approximately 68 kDa. Data shown are representative of two independent T cell samples.