Regulation of luteinizing hormone receptor expression by an RNA binding protein: role of ERK signaling

Abstract

A specific luteinizing hormone receptor (LHR) mRNA binding protein (LRBP) has been identified and purified. This LH receptor mRNA binding protein selectively binds to the polypyrimidine rich bipartite sequence in the coding region of the LHR mRNA and accelerates its degradation. In response to preovulatory LH surge, the LH receptor expression in the ovary undergoes downregulation by accelerated degradation of LH receptor mRNA through the involvement of this RNA binding protein. Here we describe the intracellular mechanism triggered by LH/hCG (human chorionic gonadotropin) that leads to the regulated degradation of LH receptor mRNA. Downregulation of LH receptor mRNA was induced by treatment of cultured human granulosa cells with 10 IU of hCG. Activation of downstream target, extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) showed an increase within five min and sustained up to 1 h. Confocal analysis showed that ERK1/2 translocates to the nucleus after 15 min of hCG treatment. This leads to an increase in LRBP expression which then causes downregulation of LH receptor mRNA by accelerating its degradation. Treatment with UO126 or transfection with ERK specific siRNA (small interfering RNA) resulted in the abolishment of ERK activation as well as LHR mRNA downregulation. RNA electrophoretic mobility gel shift assay of the cytosolic fractions showed that hCG-induced increase in the LH receptor mRNA binding activity was also abrogated by these treatments. These results show that LH/hCG-induced LH receptor mRNA downregulation is initiated by the activation of ERK1/2 pathway by regulating the expression and activity of LH receptor mRNA binding activity.

Fig. 1

Northern blot analysis of LHR mRNA in human granulosa cells immediately after retrieval (D1) and after 4 days of incubation in serum free media (D4). Total RNA was extracted from granulosa cells from day 1 and four days of incubation (D4) to recover from downregulation. RNA was separated on agarose-formaldehyde gel, transferred to nitrocellulose membranes, hybridized with the ³²P-labelled hLHRcDNA, and exposed to x-ray film. To monitor RNA loading, the blot was stripped and rehybridized with radiolabelled cDNA for 18S rRNA. The blot shown is one representative of three experiments with similar results. (Source: Ref. , Reproduced with permission).

Fig. 2

Hormonal control of LHR mRNA expression in the ovary. A. Northern blot hybridization analysis of steady state LHR mRNA levels during hCG-induced downregulation. Autoradiogram of Northern blot hybridization analysis of total RNA isolated at the indicated times from the ovaries of saline-injected (control) (lanes 1-6) or hCG-injected (downregulated) (lanes 7-11) rats. Blots were probed using a labelled cDNA encoding the LHR carboxyl terminus and a portion of the 3’-UTR (nucleotides 1936-2682). B. LHR mRNA half-life determination in control and 12 h downregulated rat ovaries. Cell suspensions were incubated with 10 µg/ml actinomycin D for 2h. Duplicate aliquots of 20 × 10⁶ cells were removed at the indicated times. Total RNA was isolated and assayed for LHR mRNA by solution hybridization. Each data point represents the average of duplicate determinations. ▀ Contol, ▲ downregulated. (Source: Ref. , Reproduced with permission).

Fig. 3

Effect of PKA inhibitor H-89 on the expression of LHR mRNA and LRBP protein in human granulosa cells. Day 3 granulosa cells were serum-starved, treated with hCG (10 IU/ml) with or without the PKA inhibitor H-89 (10 µM; 1 h pretreatment) for a total of 12 h, and were either processed for total RNA isolation or lysed using RIPA buffer. A. Total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using specific primers and probes for LHR. The graph represents changes in mRNA levels normalized to 18S rRNA, shown as fold change vs. control. Error bars, mean ± SE. *, P < 0.05 vs. control CTL; #, P < 0.05 vs. hCG; n = 4. B. Cell lysates were subjected to Western blot analysis to detect LRBP using LRBP antibody. The membranes were then stripped and reprobed for β-tubulin. The lower panel represents densitometric scanning of the LRBP normalized for tubulin and expressed as fold change vs. CTL. The blot shown is representative of three independent experiments, and the results in the bar graph are average and SE of three experiments. *, P < 0.05 vs. CTL; #, P < 0.05 vs. hCG. (Source: Ref , Reproduced with permission).

Fig. 4

Activation of ERK1/2 in human granulosa cells. Day 4 granulosa cells were serum-starved, treated with hCG (10 IU/ml) alone for different time intervals (5 min, 15 min, 30 min, and 1 h; (A) or in the presence of H-89 (10 µM; 1 h pretreatment) for 15 min (B) and were lysed using RIPA buffer. The cell lysates were subjected to Western blot analysis to detect p-ERK1/2. The membranes were then stripped and reprobed for total ERK2. Lower panels represent densitometric scanning of the p-ERK1/2 signals normalized with ERK2 and expressed as fold change vs. CTL. The blots shown are representative of three independent experiments, and the results in the bar graphs are average and SE of three experiments. *, P < 0.05 vs. CTL; #, P < 0.05 vs. hCG. (Source: Ref. , Reproduced with permission).

Fig. 5

ERK1/2 silencing inhibits hCG-induced decrease in LHR mRNA levels and increases in LRBP protein expression and binding activity. Granulosa cells were transfected with either control siRNA (CTLsi) or ERK 1/2 siRNA (ERKsi) and cultured for 48 h. After serum-starving for another 24 h, cells were treated with hCG (10 IU/ml) for 12 h and processed for total RNA isolation, for Western blot analysis, or for REMSA. A. ERK1/2 silencing was confirmed by the Western Blot analysis of cell lysates using total ERK2 antibody. B. Total RNAs were reverse transcribed, and the resulting cDNAs were subjected to real-time PCR quantitation using LHR-specific primers and probes. The graph represents changes in mRNA levels normalized to 18S rRNA and are shown as fold change vs. control. Error bars, mean ± SE.*, P < 0.05 vs. CTL; #, P < 0.05 vs. hCG; n = 3. C. Cell lysates were subjected to Western blot analysis to detect LRBP using specific antibody. The same membranes were then stripped and reprobed for ERK2 and β-tubulin. The blot shown is a representative of three independent experiments. D. G el mobility shift analysis was performed with [³²P]-labelled rat LBS (1.5 × 105 cpm) and S100 fractions containing equal amounts of total protein extracted from the different treatment groups. The autoradiogram shown is representative of three independent experiments. (Source: Ref. , Reproduced with permission).

Fig. 6

Schematic model depicting the proposed signaling pathway in LH/hCG-induced LHR mRNA down regulation. Binding of ligand to LH receptor induces activation of ERK1/2 through the cAMP/PKA pathway. This leads to an increase in the expression of LRBP and thereby its LHR mRNA binding activity, which ultimately results in LHR mRNA degradation. (Source: Ref. , Reproduced with permission).