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Am J Physiol Cell Physiol 291: C466-C475, 2006. First published April 5, 2006; doi:10.1152/ajpcell.00418.2005
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CELLULAR METABOLISM

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

Fan Zhang,1 Qimin Zhang,1 Anders Tengholm,2 and Åke Sjöholm1

1Karolinska Institute, Department of Internal Medicine, Stockholm South Hospital, Stockholm and 2Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden

Submitted 18 August 2005 ; accepted in final form 31 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that human growth hormone (hGH) increases cytoplasmic Ca2+ concentration ([Ca2+]i) and proliferation in pancreatic beta-cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca2+]i involves Ca2+-induced Ca2+ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca2+]i and insulin release in BRIN-BD11 beta-cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca2+]i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K+ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca2+]i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K+. Ovine prolactin increased [Ca2+]i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca2+]i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca2+]i. Our study indicates that hGH stimulates rise in [Ca2+]i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells.

c-Src; growth hormone receptor; prolactin receptor; Ca2+-induced Ca2+ release

GROWTH HORMONE (GH) is a major regulator of cell growth and metabolism. The actions of GH are the result of the interaction between GH and its receptors, which belong to the cytokine receptor superfamily. In rodent islet cells, effects of human (h)GH can also be mediated by the closely related prolactin (PRL) receptors (PRLRs). A significant characteristic of the cytokine receptor family is that the receptors do not contain intrinsic tyrosine kinase activity. Their transduction processes utilize nonreceptor tyrosine kinases [Janus kinases (JAKs)] and members of the signal transducers and activators of transcription (STAT) (12, 23). Interaction of GH with its receptors induces binding and activation of JAK2, leading to tyrosine phosphorylation of JAK2 itself, the GH receptor (GHR), as well as other target proteins. Studies using truncated and mutated GHRs have shown that the box 1 region is necessary for association and activation of JAK2 (10). Activation of JAK2 is believed to be critical for initiation of most, if not all, GH-induced signaling pathways (8, 21, 25, 28, 40, 59). In addition to JAK2, the Src tyrosine kinase family has been shown to be involved in GH signaling (13, 61).

The pivotal role of Ca2+ in insulin secretion is well documented (22, 37, 58). Influx of Ca2+ through voltage-gated channels in the plasma membrane is the main trigger of exocytosis of insulin secretory granules (22). In addition, intracellular Ca2+ stores play a significant role in Ca2+ homeostasis and regulation of multiple cellular functions. The endoplasmic reticulum represents the major Ca2+ store in the pancreatic beta-cell (31, 50, 51, 56), but the Ca2+-rich dense core secretory vesicles have also been proposed to form a dynamic Ca2+ store in this cell type (39). The intracellular Ca2+ stores can be mobilized through activation of inositol 1,4,5-trisphosphate (InsP3) receptors and ryanodine receptors (RyRs). Both types of receptors can be gated by Ca2+ itself (62), resulting in release of Ca2+, a process called Ca2+-induced Ca2+ release (CICR). This process has been described in different cell types, including insulin-secreting cells (7, 11, 16, 26). However, it is unknown to what extent CICR participates in the regulation of insulin secretion.

CICR via RyRs is regulated by phosphorylation of the intracellular Ca2+ release channels, leading to increased channel opening (19, 24, 33). We showed previously that hGH stimulation of beta-cell proliferation is associated with an increase of the cytoplasmic Ca2+ concentration ([Ca2+]i) (43) and that the hGH-induced elevation of [Ca2+]i involves tyrosine phosphorylation of RyRs in BRIN-BD11 beta-cells (60). The latter study suggests that tyrosine kinases play an important role in activation of CICR by hGH in insulin-secreting cells. In the present study, we investigated the involvement of JAK2 and Src tyrosine kinases in hGH-induced rise in [Ca2+]i and insulin secretion in the BRIN-BD11 beta-cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Recombinant hGH was graciously donated by Pharmacia (Stockholm, Sweden). Bovine (b)GH and ovine (o)PRL were from Biogenesis (Poole, UK). Tyrphostin B42 (AG490), PP2, PP3, JAK2 inhibitor II (1,2,3,4,5,6-hexabromocyclohexane), and Src kinase inhibitor I [4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline] were from Calbiochem (La Jolla, CA). Fura-2 acetoxymethyl ester (AM) was from Sigma (St. Louis, MO). Anti-phospho-JAK2 (Tyr 1007/Tyr 1008), anti-JAK2, anti-c-Src (N-16, H-12), horseradish peroxidase-labeled goat anti-rabbit IgG, and protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody was from Zymed (South San Francisco, CA), and protease inhibitor cocktail was from Roche Diagnostics (Mannheim, Germany). Re-blot plus strong solution was from Chemicon (Temecula, CA). The enhanced chemiluminescence (ECL) kit was from Amersham Biosciences (Chalfont St. Giles, UK). The rat insulin ELISA kit was from Mercodia (Uppsala, Sweden). Culture medium RPMI 1640 and fetal bovine serum (FBS) were purchased from Life Technologies Invitrogen (Paisley, UK).

Cell culture. Insulin-secreting BRIN-BD11 beta-cells, passages 35–50, were cultured in RPMI 1640 tissue culture medium containing 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The BRIN-BD11 beta-cell line was established by electrofusion of RINm5F insulinoma cells with New England Deaconess Hospital (NEDH) rat pancreatic islet cells and shares the major features of normal pancreatic beta-cells (35, 36).

Immunoprecipitation and Western blot analysis. BRIN-BD11 beta-cells were incubated in serum-free RPMI 1640 medium containing 1% BSA for 24 h at 37°C. Cells were then incubated with AG490 (30 µM) or PP2 (10 µM) for 10 min at 37°C in buffer A, containing (mM) 125 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, and 25 HEPES, with 0.1% BSA, pH 7.4, in the presence of 3 mM glucose, while vehicle (DMSO) was present in controls. Cells were further incubated at 37°C for 2 or 5 min in the presence or absence of GH. At the end of the incubation, cells were immediately transferred on ice and collected by a brief centrifugation. After being washed once with ice-cold PBS, cells were solubilized in a lysis buffer containing 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1x protease inhibitor cocktail, and 1% Triton X-100 in PBS, pH 7.5 (42), on ice for 40 min. The cell lysate was clarified by centrifugation (10,000 g, 10 min, 4°C), and the supernatant was collected and normalized with a protein assay (Bio-Rad). For Western blot analysis, cell lysates containing equal amounts of protein (40 µg) were mixed with SDS-sample buffer and subjected to SDS-PAGE under reducing conditions. Proteins in the gel were subsequently electrotransferred onto nitrocellulose membranes. The membrane was blocked in 20 mM Tris base and 137 mM NaCl, pH 7.6 with 0.05% Tween 20 (TBS-T) with 5% nonfat dry milk, followed by an overnight incubation with anti-phospho-JAK2 (1:200) in TBS-T-1% BSA at 4°C. After extensive washes in TBS-T, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:10,000) in TBS-T-1% BSA for 1 h at room temperature. The membrane was extensively washed, and the immunostained proteins were visualized by ECL. For detection of phosphorylated c-Src, lysates containing equal amounts of protein (200 µg) were incubated with anti-c-Src (H-12; 2 µg/ml) overnight with gentle shaking at 4°C. The immunocomplex was precipitated with protein A/G plus agarose (4°C, 2 h). After the beads were washed three times with lysis buffer, the immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions. After protein transfer, the membrane was probed by anti-phosphotyrosine antibody (1:2,000) overnight at 4°C, followed by incubation with horseradish peroxidase-labeled secondary antibody. The immunostained proteins were visualized by ECL. The blots were stripped in Re-blot plus strong solution and probed with either anti-JAK2 (1:200) or anti-c-Src (N-16; 1:400) antibodies. The intensities of the bands thus obtained were quantified by densitometry.

Measurement of [Ca2+]i. BRIN-BD11 beta-cells grown on glass coverslips were loaded with fura-2 (1.5 µM) for 30 min at 37°C in buffer A in the presence of 3 mM glucose. Where indicated, cells were further incubated with AG490 (30 µM), PP2 (10 µM), or vehicle (DMSO) for 10 min. The effect of JAK2 inhibitor II or Src kinase inhibitor-1 on hormone- or K+-induced rise in [Ca2+]i was investigated by preincubation of the cells with inhibitors or vehicle for 16 h in an incubator. The coverslips with cells were subsequently rinsed once in the same buffer without the Ca2+ indicator and mounted as the bottom of a perifusion chamber on the stage of an inverted microscope (Nikon Diaphot). The stage was thermostated to 37°C, and the cells were superfused at a rate of 300 µl/min with buffer A containing 3 mM glucose. Measurements of [Ca2+]i were performed as previously described (30) with a time-sharing spectrofluorometer providing light flashes of 1-ms duration at 340 nm/380 nm every 10 ms. Fluorescence was recorded at 510 nm from small clusters of three to five cells. [Ca2+]i was calculated from the 340 nm/380 nm fluorescence excitation ratio according to Grynkiewicz et al. (17). Cells were perifused in the presence or absence of the inhibitors before and during the first stimulation with hormone or high K+ concentration. The inhibitors were subsequently withdrawn, and the responses of the cells to hormone or K+ were reexamined. In the case of application of JAK2 inhibitor II or Src kinase inhibitor-1, hormone responses of the cells preincubated with vehicle alone acted as controls.

Insulin secretion. BRIN-BD11 beta-cells were collected and washed three times in buffer A containing 0.1% BSA. Equal amounts of cells were placed in 48-well plates. Incubation was performed in the same buffer containing 3 mM glucose in the presence of AG490 (30 µM), PP2 (10 µM), or vehicle (DMSO) at 37°C for 10 min, followed by stimulation with hormone or K+ at 37°C for 20 min. The effect of JAK2 inhibitor II or Src kinase inhibitor-1 on hormone- or K+-induced insulin secretion was investigated by preincubation of the cells with inhibitors or vehicle for 16 h in an incubator. Cells were collected and washed as above. Incubation was carried out in the buffer containing indicated concentrations of the inhibitors in the presence or absence of hormone or K+ for 20 min at 37°C. At the end of incubation, supernatants were collected after centrifugation and the insulin content was measured by ELISA.

Statistical analysis. Statistical significance was tested with ANOVA as indicated in Figs. 1–5 and 7.


Figure 1
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  		Fig. 1. Rapid human growth hormone (hGH)-stimulated tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src. Quiescent BRIN-BD11 beta-cells were pretreated with vehicle or AG490 (30 µM) for 10 min at 37°C, followed by incubation with or without hGH (25 nM) for 2 (lanes 1, 2, 4) or 5 (lane 3) min at 37°C. Western blot analysis was performed with anti-phospho-JAK2 (A, top). A, bottom, shows the same blot probed with anti-JAK2 after stripping. In B, cells were prepared as in A except that they were treated with PP2 (10 µM) instead of AG490. Cells were solubilized after incubation and immunoprecipitated with anti-c-Src, followed by Western blot analysis using anti-phosphotyrosine antibody (top). B, bottom, shows the same blot probed with anti-c-Src after stripping. Representatives of 3 separate experiments for each are shown. In C and D, band densities of tyrosine phosphorylated JAK2 and c-Src were measured and normalized by total JAK2 or c-Src proteins obtained after reblotting with anti-JAK2 or c-Src antibodies. The results are expressed as % control (with vehicle). Means ± SE derived from 3 separate experiments for each are shown (*P < 0.05 for a chance difference by ANOVA).

 

Figure 5
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  		Fig. 5. Effect of ovine prolactin (oPRL) on [Ca2+]i in BRIN-BD11 beta-cells. Experiments were performed and analyzed as described in Fig. 2. oPRL (25 nM) was applied as indicated. Cells were treated with the inhibitors as described in Fig. 2. In the case of JAK2 inhibitor II or Src kinase inhibitor-1, hormone responses in cells preincubated with vehicle alone acted as controls. Representative traces of 7 (A, B, and D), or 6 (C and E) are shown. F shows the summary data (means ± SE) derived from the experiments performed (*P < 0.05, compared with control, ANOVA).

 

Figure 7
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  		Fig. 7. Effect of JAK2 and Src kinase inhibitors on insulin secretion stimulated by oPRL and requirement of Ca2+ in hGH-induced insulin secretion. oPRL-induced insulin secretion was examined as in Fig. 4 and is shown in A and B. The effect of bGH on insulin secretion, compared to hGH (C), was evaluated after 20-min incubation as described in Fig. 4. D and E show hGH-induced insulin secretion in the presence or absence of extracellular Ca2+ (without Ca2+ addition in the buffer and in the presence of 2 mM EGTA) or after the cells were pretreated with thapsigargin (250 nM, 30 min), respectively. Insulin secretion is expressed as % changes compared to the corresponding controls. (Conditions with Ca2+-containing and Ca2+-free buffers without hormone are controls of the hormone effect in the presence or absence of Ca2+, respectively. A similar comparison was performed in thapsigargin analysis). Means ± SE derived from 5 (A), 6 (B), or 4 (C–E) separate experiments are shown (*P < 0.05, ANOVA).

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
hGH rapidly stimulates tyrosine phosphorylation of JAK2 and c-Src. As hGH-stimulated rise in [Ca2+]i is tyrosine kinase dependent (60) and JAK2 and Src tyrosine kinases are known to be involved in GH signaling, we first investigated the effect of hGH on activation of JAK2 and c-Src in the cells. As shown in Fig. 1A, hGH (25 nM) markedly stimulated tyrosine phosphorylation of JAK2, confirming previous observations in pancreatic beta-cells (9). The phosphorylation of JAK2 was maximal at 2 min after hGH addition and decreased thereafter. To investigate the specificity of the hGH-stimulated phosphorylation of JAK2, we applied the JAK2 inhibitor AG490, whose specificity is well documented (1, 32, 34, 38, 61). Importantly, AG490 does not affect hGH-stimulated Src activation (38, 61). Pretreatment of the cells with the inhibitor at 30 µM suppressed hGH-induced phosphorylation of JAK2 (Fig. 1A) to levels not significantly different from controls (Fig. 1C).

The ability of hGH to activate c-Src was examined after 2-min stimulation with the hormone at 25 nM. As shown in Fig. 1, B and D, hGH induced a robust tyrosine phosphorylation of c-Src. This effect was abolished by 10 µM PP2, a specific inhibitor of Src kinases (20, 48, 57, 61), which has been shown to be without effect on JAK2 activation induced by hGH (20, 61).

hGH-induced increase in [Ca2+]i is blocked by inhibition of JAK2 and c-Src activation. We next investigated whether the hGH-stimulated rise in [Ca2+]i was affected by inhibition of JAK2 and Src tyrosine kinases. hGH (25 nM) elicited a rapid and robust rise in [Ca2+]i in BRIN-BD11 beta-cells (Fig. 2A), consistent with previous findings (60). Pretreatment of the cells with AG490 (30 µM) resulted in a slight reduction of basal [Ca2+]i and a nearly complete inhibition of the hGH effect (Fig. 2, B and F). The effects were reversible, and after withdrawal of AG490 both basal and hGH-stimulated [Ca2+]i were fully normalized. In addition, another JAK2 kinase inhibitor, JAK2 inhibitor II, a small-molecule inhibitor of JAK2 tyrosine kinase (43), was applied. In control experiments in which cells were preincubated with vehicle, hGH evoked a rise in [Ca2+]i to the same extent as in Fig. 2A. In contrast, JAK2 inhibitor II, at a concentration of 1 µM, almost abolished the hGH-induced rise in [Ca2+]i, although the cells readily responded to high K+ (Fig. 2, C and F).


Figure 2
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  		Fig. 2. Effect of JAK2 and Src kinase inhibitors on hGH-induced increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in BRIN-BD11 beta-cells. After fura-2 loading, cells were perifused with buffer A containing 3 mM glucose. Additions of hGH (25 nM) are indicated. Cells were preincubated with AG490 (AG, 30 µM; B), PP2 (10 µM; D), PP3 (10 µM), or vehicle for 10 min, and the inhibitors were present before and during the first addition of hGH. In C and E, cells were pretreated with JAK2 inhibitor II (JAK2-II, 1 µM), Src kinase inhibitor-1 (Src-I, 5 µM) or vehicle (as control) for 16 h in an incubator and experiments were performed as in B. Hormone responses in experiments in which cells were preincubated with vehicle acted as controls. The inhibitors were continuously present during perifusion as indicated. Cells were depolarized by K+ (25 mM) at the end of the experiments, serving as a control for cell responsiveness. Representative traces of 7 (A and D), 7 (B), or 6 (C and E) experiments are shown. The inhibitory effect of the JAK2 or Src kinase inhibitors on hGH-induced rise in [Ca2+]i is summarized in F and represented as % inhibition (means ± SE; *P < 0.05, compared with hGH response by ANOVA).

 
Preincubation of cells with PP2 (10 µM) reduced the hGH-induced [Ca2+]i response by ~50% without any significant changes in the initial rate or duration of the [Ca2+]i elevation (Fig. 2, D and F). Increasing the PP2 concentration to 30 µM did not cause further inhibition (data not shown). In contrast, the inactive analog PP3 had no effect on hGH-induced [Ca2+]i increase (Fig. 2F). After preincubation of the cells with 5 µM Src kinase inhibitor-1, another specific Src kinase inhibitor (53), the effect of hGH was nearly completely inhibited (Fig. 2, E and F). No significant changes in cell viability were observed after 16-h incubation with up to 10 µM of either JAK2 inhibitor II or Src kinase inhibitor-1 as evaluated by Trypan blue exclusion.

The specificity of the tyrosine kinase inhibitors for hGH-induced rise in [Ca2+]i was evaluated by depolarizing BRIN-BD11 beta-cells with a high concentration of K+. The cells were stimulated by two pulses of K+ (25 mM), which both induced large increases in [Ca2+]i, although the second response was often somewhat smaller than the first (Fig. 3A). Whereas PP2 was without effect (Fig. 3, D and E), AG490 reduced the K+-induced rise in [Ca2+]i by ~50% (Fig. 3, B and E). However, this inhibitory effect was no longer observed after depletion of intracellular Ca2+ stores by pretreatment with the Ca2+-ATPase inhibitor thapsigargin (250 nM) (Fig. 3, C and E). At the dosages that inhibited hGH-induced rise in [Ca2+]i, neither JAK2 inhibitor II nor Src kinase inhibitor-1 significantly influenced the K+-stimulated rise in [Ca2+]i (Fig. 3F).


Figure 3
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  		Fig. 3. Effect of JAK2 or Src kinase inhibitors on K+-induced rise in [Ca2+]i in BRIN-BD11 beta-cells. Cells were preincubated with vehicle (A), AG490 (B and C), PP2 (D), and JAK2 inhibitor II or Src kinase inhibitor-1 (F) as described in Fig. 2. Stimulation of the cells with K+ (25 mM) is indicated. In C, cells were preincubated with the Ca2+-ATPase inhibitor thapsigargin (250 nM) for 30 min during loading of the cells with fura-2. Cells were further incubated with AG490 (30 µM) or vehicle for 10 min before measurement. E shows the ratios of the first peak (P1) stimulated by K+ over the second (P2), derived from 4 separate experiments for each under the conditions as described in A–D (means ± SE; *P < 0.05 for a chance difference by ANOVA). Experiments similar to those in A–D were performed with cells preincubated for 16 h with JAK2 inhibitor II, Src kinase inhibitor-1, or vehicle (controls), and the data are summarized in F. The changes in ratios of the first over the second Ca2+ peaks induced by K+ and derived from 6 separate experiments are shown.

 
hGH-induced insulin secretion is blocked by inhibition of JAK2 and c-Src activation. To investigate whether the hGH-stimulated rise in [Ca2+]i translated into changes in exocytosis, insulin secretion was examined after incubation of the cells with hGH for 20 min. hGH increased insulin secretion by ~75% (Fig. 4A), which is similar to the response induced by glucose (Fig. 4D). The effect of hGH was completely abolished by pretreatment of the cells with 30 µM AG490 (Fig. 4A) or 10 µM PP2, but not the inactive analog PP3 (Fig. 4B). AG490, but not PP2, also caused a slight but significant suppression of basal insulin release. Application of JAK2 inhibitor II or Src kinase inhibitor-1 resulted in a dose-dependent inhibition of insulin secretion evoked by hGH. One micromolar JAK2 inhibitor-II or five micromolar Src kinase inhibitor-1 completely abolished the hGH-induced release of insulin without interfering with basal secretion (Fig. 4C). In contrast to their effects on insulin secretion evoked by hGH, none of the tyrosine kinase inhibitors significantly affected insulin secretion triggered by 25 mM K+ (Fig. 4E).


Figure 4
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  		Fig. 4. Effect of JAK2 and Src kinase inhibitors on hGH- and K+-induced insulin secretion in BRIN-BD11 beta-cells. In A, cells were preincubated with AG490 (30 µM) or vehicle for 10 min, followed by stimulation of the cells with hGH (25 nM) for 20 min at 37°C as indicated. In B, instead of AG490, cells were preincubated with PP2 (10 µM), PP3, or vehicle for 10 min and experiments were performed as in A. The effect of JAK2 inhibitor II or Src kinase inhibitor-1 on hGH-induced insulin secretion was investigated after preincubation of the cells with indicated concentrations of the inhibitors or vehicle for 16 h (C). The inhibitors were present during the 20-min incubation in the presence or absence of hGH. Glucose-stimulated insulin secretion from the cells was examined after incubation of the cells in the presence of indicated concentrations of glucose for 20 min, and the result is expressed as % increase compared with insulin secretion at 1.1 mM glucose (D). In E, experiments were performed as in A–C. Instead of hGH, cells were stimulated with K+ (25 mM) for 20 min after preincubation with the inhibitors as indicated. Means ± SE derived from 5 (A and B), 5 (C), or 3 (D and E) separate experiments are shown and are expressed as % changes compared to controls (*P < 0.05, ANOVA). The chance difference comparison groups are indicated in A and B. In C, D, and E, significant changes compared with controls are shown.

 
oPRL, but not bGH, mimicked effects of hGH on [Ca2+]i and insulin secretion. Because hGH is known to interact not only with the GHR but also with the PRLR, both of which are present in rat insulin-secreting beta-cells (6, 14, 44), we applied oPRL and bGH, which interact only with the PRLR and the GHR (15), respectively. Application of oPRL at the same dosage as hGH (25 nM) stimulated a rise in [Ca2+]i to a similar extent as hGH (Fig. 5A). The effect of oPRL was nearly completely abolished by either the JAK2 inhibitors AG490 (Fig. 5, B and F) and JAK2 inhibitor II (Fig. 5, C and F) or the Src kinase inhibitors PP2 (Fig. 5, D and F) and Src kinase inhibitor-1 (Fig. 5, E and F), whereas in control experiments in which cells were preincubated with vehicle for 16 h, oPRL evoked a rise in [Ca2+]i to the same extent as in Fig. 5A. Stimulation of the cells with bGH also resulted in a rise in [Ca2+]i (Fig. 6A). However, neither the JAK2 inhibitors AG490 (Fig. 6B) and JAK2 inhibitor II (Fig. 6C) nor the Src kinase inhibitors PP2 (Fig. 6D) and Src kinase inhibitor-1 (Fig. 6E) interfered with the effect of bGH.


Figure 6
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  		Fig. 6. Effect of bovine (b)GH on [Ca2+]i in BRIN-BD11 beta-cells. Experiments similar to those in Fig. 2 were performed. bGH (25 nM) was applied as indicated. Representative traces of 6 (A and D), 7 (B), or 5 (C and E) are shown.

 
The effect of oPRL on insulin secretion from the cells was evaluated under the same conditions as hGH. Stimulation of the cells with 25 nM oPRL for 20 min resulted in insulin secretion to a similar extent as hGH (Fig. 7A). oPRL-stimulated insulin secretion was completely inhibited by AG490 or PP2, but not by the inactive analog PP3 (Fig. 7A). In addition, at the concentrations of 1 µM JAK2 inhibitor II and 5 µM Src kinase inhibitor-1, the effect of oPRL was completely abolished (Fig. 7B). In contrast, bGH, at the same concentration as hGH or oPRL, did not show any effect on insulin secretion (Fig. 7C).

To clarify whether elevation of [Ca2+]i is required for hGH-induced insulin secretion, cells were incubated in a Ca2+-free medium in the presence of EGTA. Under these conditions, hGH failed to stimulate insulin secretion (Fig. 7D). In addition, pretreatment of the cells with thapsigargin to empty cytosolic Ca2+ pools prevented the effect of hGH (Fig. 7E).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We recently demonstrated (60) that the hGH-stimulated rise in [Ca2+]i involves tyrosine phosphorylation of RyRs in insulin-secreting BRIN-BD11 beta-cells. The present study shows that hGH stimulated rapid tyrosine phosphorylation of JAK2 and c-Src tyrosine kinases, which is associated with increased [Ca2+]i and insulin secretion in this cell type. The effect of hGH on [Ca2+]i and insulin secretion was mimicked by oPRL, but not by bGH, indicating that its effects are conveyed through the PRLR.

Activation of JAK2 is essential for initiation of many biological actions of GH in cells (2, 46). In pancreatic beta-cells, GH-mediated proliferation is directly mediated via JAK2 activation (9). The present study in insulin-secreting BRIN-BD11 beta-cells shows that blockade of JAK2 activation inhibits hGH-induced tyrosine phosphorylation of JAK2 and the effect of hGH on both [Ca2+]i and insulin secretion, suggesting an essential role of JAK2 activation in these events. The time course of JAK2 tyrosine phosphorylation observed in the hGH-stimulated BRIN-BD11 beta-cells is similar to that previously found for tyrosine phosphorylation of RyR, where a marked phosphorylation of the receptor was observed after 2-min stimulation with the hormone (60). Because hGH-induced rise in [Ca2+]i involves tyrosine phosphorylation of RyR (60) and the effect of hGH on [Ca2+]i was abolished when GH-stimulated JAK2 phosphorylation was inhibited by specific JAK2 inhibitors in the present study, it is conceivable that hGH induces a direct interaction between JAK2 and RyR.

The JAK2-dependent manner by which hGH induced a rise in [Ca2+]i in the insulin-secreting cells seems fundamentally different from that in Chinese hamster ovary (CHO) cells transfected with the GHR (4). In the transfected CHO cells, mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding and activation of JAK2 kinase, failed to affect the GH-induced rise in [Ca2+]i despite a complete inhibition on GH-induced gene transcription. This apparent discrepancy may be due to the fact that hGH mainly acts through the PRLR in rodent islets (4, 5). Accordingly, the increase in [Ca2+]i induced by activation of CHO cells transfected with PRLR requires activation of JAK2 and tyrosine phosphorylation (41, 45). The JAK2-independent pattern of [Ca2+]i increase, mediated through the GH receptor, suggests that rise in [Ca2+]i may not be directly involved in the rat GH-stimulated proliferation in pancreatic beta-cells (9). The similarities in the functional responses of hGH and oPRL in the present study indicate that the PRLR acts as the major player in the hGH actions studied here. The inability of hGH to stimulate insulin secretion in the absence of extracellular Ca2+ or after emptying of the intracellular Ca2+ pools with thapsigargin indicates that elevation of [Ca2+]i is required for hGH-evoked insulin release. Interestingly, our results imply that a rise in [Ca2+]i elicited by PRLR agonists is sensitive to JAK2 inhibitors and associated with enhanced insulin secretion in BRIN-BD11 beta-cells. In contrast, an increase in [Ca2+]i through the GHR is insensitive to the inhibitors and is dissociated from insulin secretion.

In addition to its inhibitory effect on hGH-induced rise in [Ca2+]i, AG490, but not JAK2 inhibitor II, was also shown to slightly lower the resting [Ca2+]i and to attenuate K+-stimulated rise in [Ca2+]i. The latter effect was not observed after depletion of the intracellular Ca2+ stores, indicating that AG490 does not interfere with Ca2+ entry through the voltage-gated Ca2+ channels but rather with intracellular Ca2+ mobilization that may be triggered by the depolarization and Ca2+ influx (52). However, it appears unlikely that nonspecific effects of AG490 could account for the complete inhibition of the hGH- and oPRL-induced rise in [Ca2+]i and insulin secretion. First, AG490 had no effect on bGH-stimulated increase in [Ca2+]i and K+-induced insulin secretion. Second, JAK2 inhibitor II mimicked the inhibitory actions of AG490 without nonspecific effects on the K+-stimulated rise of [Ca2+]i.

In addition to JAK2, stimulation of BRIN-BD11 beta-cells with hGH resulted in activation of c-Src, which has been shown to be involved in GH signaling in other systems (29, 61) and to participate in cell signaling also in insulin-secreting cells (49). That the phosphorylation of c-Src stimulated by hGH was completely inhibited by PP2 indicates an activating tyrosine phosphorylation induced by the hormone. Additionally, the enhanced rise in [Ca2+]i and insulin secretion by either hGH or oPRL were prevented by two different Src kinase inhibitors, whereas PP3 had no effect, suggesting a requirement of activation of Src kinases in the biological actions. In contrast to their effect on hGH- and oPRL-stimulated events, neither PP2 nor Src kinase inhibitor-1 had any effect on bGH-stimulated rise in [Ca2+]i, indicating that the cellular events mediated through the PRLR, but not through the GHR, require activation of Src kinases. Among Src kinases, c-Src is abundantly expressed in tissues and has been shown to be involved in Ca2+ mobilization (47, 54). However, we cannot exclude an involvement of additional members of the Src kinase family in hGH actions. For example, the tyrosine kinase fyn has been implicated in RyR phosphorylation and Ca2+ mobilization in T cells (18).

The complete inhibition of hGH-stimulated insulin secretion by PP2 may appear surprising, because the Src kinase inhibitor only partially suppressed the hGH-induced rise in [Ca2+]i. However, although insulin release is Ca2+ dependent, Ca2+ may not be the only determinant for secretion. Interestingly, several synaptic proteins involved in vesicle trafficking and exocytosis, such as synaptophysin, synaptogyrin, and cellugyrin, have been found to be substrates for c-Src (3, 27, 55). Further experiments are required to elucidate the precise role of the Src family kinases and the activation sites of the Src kinases in the hGH-induced rise of [Ca2+]i and insulin secretion.

In conclusion, the present study shows that hGH rapidly stimulates tyrosine phosphorylation of both JAK2 and c-Src, which is mediated mainly through the PRLR. This mechanism may be involved in the tyrosine kinase-dependent increase in [Ca2+]i and insulin secretion elicited by the hormone.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
Financial support was received from the Swedish Medical Research Council (nos. 72X-12550, 72X-14507, and 72P-14787), Berth von Kantzow’s Foundation, SmithKline Beecham Pharma AB, Petrus and Augusta Hedlund’s Foundation, the Nutricia Research Foundation, the European Foundation for the Study of Diabetes, the Swedish Society of Medicine, the Sigurd and Elsa Golje Memorial Foundation, Svenska Försäkringsföreningen, the Novo Nordisk Foundation, Svenska Diabetesstiftelsen, Magn. Bergvall Foundation, Novo-Nordisk Sweden Pharma AB, Barndiabetesfonden, Åke Wiberg’s Foundation, Torsten and Ragnar Söderberg’s Foundations, Harald Jeansson’s and Harald and Greta Jeansson’s Foundations, Tore Nilson’s Foundation for Medical Research, Fredrik and Inger Thuring’s Foundation, Syskonen Svensson’s Fund (all to Å. Sjöholm) and Swedish Research Council 32XD-14643, European Foundation for the Study of Diabetes/Novo Nordisk, Novo Nordisk Foundation, the Family Ernfors Foundation, and Wenner-Gren Foundations (to A. Tengholm).


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Zhang, Research Ctr., Karolinska Inst., Stockholm South Hospital, SE-11883 Stockholm, Sweden (e-mail: qimin.zhang{at}sos.ki.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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