RANK Ligand-induced Elevation of Cytosolic Calcium Accelerates Nuclear Translocation of Nuclear Factor B in Osteoclasts

Experimental Design:

Osteoclast Isolation and Culture: Osteoclasts were isolated from neonatal rats and rabbits. Various single-cell techniques like microspectrofluorimetry were employed to monitor cytosolic calcium levels ([Ca²⁺]i), while patch clamp methods were used to assess membrane currents. Immunofluorescence was used to visualize NFκB translocation in osteoclasts.
Test Substances:
- Soluble recombinant RANKL was applied to the osteoclasts.
- Various inhibitors were used to modulate the signaling pathways:
  - U73122 (phospholipase C inhibitor)
  - BAPTA-AM (calcium chelator)
  - Cyclosporin A and FK506 (calcineurin inhibitors)
  - Bisindolylmaleimide I (PKC inhibitor)
  - Control compounds like U73343 and Bisindolylmaleimide V, which do not inhibit PLC or PKC, respectively, were used to confirm the specificity of the inhibitors.

Results:

RANKL Induces Elevation of Cytosolic Ca²⁺:
- RANKL increased cytosolic Ca²⁺ in osteoclasts, with an average increase of 220 nM above basal levels. This increase was transient, returning to baseline after RANKL washout.
- RANKL-induced Ca²⁺ transients were observed in both Ca²⁺-containing and Ca²⁺-free media, indicating that the Ca²⁺ was released from intracellular stores.
- The phospholipase C (PLC) inhibitor U73122 abolished RANKL-induced Ca²⁺ elevation, while the control compound U73343 had no effect. This confirms that the Ca²⁺ release was mediated by the PLC pathway.
RANKL Activates Ca²⁺-Dependent K⁺ Currents:
- Using patch clamp techniques, RANKL was shown to activate intermediate conductance Ca²⁺-dependent potassium (K⁺) channels in rabbit osteoclasts. These channels were activated following the rise in [Ca²⁺]i, which further confirms that RANKL induces intracellular Ca²⁺ signaling.
Calcineurin and Protein Kinase C (PKC) Regulate NFκB Translocation:
- Calcineurin's Role: Inhibition of calcineurin with cyclosporin A or FK506 delayed NFκB translocation from the cytoplasm to the nucleus. The effect was most pronounced at the early stages (7 minutes after RANKL stimulation), but the inhibition had no effect on NFκB translocation at later time points (15-30 minutes). This indicates that calcineurin, activated by Ca²⁺, plays a key role in the early acceleration of NFκB translocation.
- PKC's Role: The PKC inhibitor bisindolylmaleimide I also delayed NFκB translocation. Unlike calcineurin inhibition, PKC inhibition affected both the early and intermediate time points (7 and 15 minutes), suggesting that PKC activity is important not only for the initiation but also for sustaining NFκB nuclear translocation. The effects of PKC inhibition were additive to those of calcineurin inhibitors, indicating that both pathways independently contribute to the regulation of NFκB translocation speed.
Effect of BAPTA on NFκB Translocation:
- Loading osteoclasts with the Ca²⁺ chelator BAPTA-AM delayed NFκB nuclear translocation following RANKL treatment. Maximum translocation was observed at 30-60 minutes in BAPTA-treated cells, compared to 15-30 minutes in control cells treated with RANKL alone. This suggests that the elevation of cytosolic Ca²⁺ is crucial for accelerating NFκB activation.
- BAPTA also reduced the overall number of osteoclasts exhibiting NFκB nuclear translocation, confirming the importance of Ca²⁺ in the process. In contrast, a control compound (calcein blue-AM) did not affect NFκB translocation, ruling out nonspecific effects of the loading procedure.
Ca²⁺ is Necessary for Osteoclast Survival:
- RANKL significantly increased the survival of osteoclasts after 24 hours of culture. However, when intracellular Ca²⁺ was chelated using BAPTA-AM, RANKL failed to promote osteoclast survival. This suggests that Ca²⁺ elevation is necessary for the pro-survival effects of RANKL in osteoclasts.
PLC Inhibition Slows NFκB Translocation:
- The PLC inhibitor U73122 delayed NFκB translocation, shifting the peak from 15 minutes to 30 minutes after RANKL addition. However, U73122 did not affect the overall proportion of osteoclasts exhibiting NFκB translocation. This indicates that while PLC-mediated Ca²⁺ signaling affects the speed of NFκB activation, it does not completely prevent it.

Implications:

The study demonstrates that RANKL signaling in osteoclasts involves the release of Ca²⁺ from intracellular stores via PLC, which accelerates NFκB translocation and promotes osteoclast survival.
Calcineurin and PKC are key downstream effectors that regulate the timing of NFκB translocation, with calcineurin affecting the initial phase and PKC playing a more sustained role.
The findings suggest that targeting the Ca²⁺ signaling pathway could modulate NFκB activity in osteoclasts, providing potential therapeutic approaches for bone diseases like osteoporosis, where osteoclast activity is dysregulated.

Core Bone Remodeling Equations

RANKL-Induced Activation of Osteoclasts via RANK and PLC Pathway:

RANKL + RANK \to Activation of PLC \to {Production of IP}_{3} \to {Release of Ca}^{2 +} from ER \to↑ [{Ca}^{2 +}]_{i}

Elevation of Cytosolic Ca²⁺ Activates Calcineurin and PKC:

\begin{matrix} ↑ [{Ca}^{2 +}]_{i} \to {\begin{cases} Activation of Calcineurin \\ Activation of PKC \end{cases} \end{matrix}

Calcineurin and PKC Facilitate NFκB Nuclear Translocation:

{\begin{cases} Calcineurin \to Dephosphorylation of NF κ B \\ PKC \to Phosphorylation of I κ B  Kinase (IKK) \to Degradation of I κ B \end{cases} \to NF κ B Translocation to Nucleus

NFκB Activation Leads to Osteoclast Survival and Activity:

NF κ B in Nucleus \to Gene Transcription \to Osteoclast Survival and Activation

Role of Calcium Chelators and Inhibitors:

BAPTA-AM (Calcium Chelator) Reduces [Ca²⁺]₍i₎ and Osteoclast Survival:

BAPTA-AM \to↓ [{Ca}^{2 +}]_{i} \to Delayed NF κ B Translocation \to Reduced Osteoclast Survival

PLC Inhibitor (U73122) Slows NFκB Translocation:

U73122 \to Inhibition of PLC \to {Reduced IP}_{3} \to {Reduced Ca}^{2 +} Release \to Delayed NF κ B Activation

Calcineurin and PKC Inhibitors Delay NFκB Activation:

Calcineurin Inhibitors (Cyclosporin A, FK506):

Cyclosporin A / FK506 \to Inhibition of Calcineurin \to Delayed NF κ B Translocation

PKC Inhibitor (Bisindolylmaleimide I):

Bisindolylmaleimide I \to Inhibition of PKC \to Delayed and Sustained NF κ B Activation

Overall Pathway Summarized:

RANKL + RANK \to PLC Activation \to {IP}_{3} \to↑ [{Ca}^{2 +}]_{i} \to Activation of Calcineurin and PKC \to NF κ B Translocation \to Osteoclast Activation and Survival

Negative Regulation by Osteoprotegerin (OPG):

OPG + RANKL \to Inhibition of RANK-RANKL Interaction \to Reduced Osteoclast Activation

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

Background: T-cell activation is crucial for adaptive immune responses, requiring calcium influx to initiate intracellular signaling cascades, including the activation of NFAT and the production of IL-2. This study explores how ATP release, acting in an autocrine manner through P2X7 receptors, regulates these key processes in T-cell activation.

Experimental Design:

The authors utilized a variety of techniques, including real-time PCR, immunohistochemistry, fluorescence microscopy, ELISA, and flow cytometry, to examine:

Jurkat T cells and human CD4+ T cells.
Mouse models (BALB/c mice with functional P2X7 receptors and C57BL/6 mice with defective P2X7 receptors) to investigate the role of P2X7 receptors in T-cell function.
Manipulations of ATP levels (via ATP scavengers) and siRNA to silence P2X7 receptors.
Pharmacological inhibitors of P2 receptors (e.g., suramin, NF023, o-ATP).

Key Results and Important Details:

ATP Release and T-Cell Activation:
- TCR and CD28 stimulation of Jurkat T cells leads to a rapid release of ATP, with extracellular ATP levels peaking near the cell surface at around 60 μM within 2 minutes. These ATP concentrations are sufficient to stimulate P2X receptors but are below the threshold that induces apoptosis (>100 μM).
- ATP release continues for up to 5 minutes, indicating its sustained role during early activation stages.
Role of ATP in Calcium Signaling:
- ATP release is required for calcium influx following TCR activation. Scavenging ATP using apyrase or alkaline phosphatase significantly inhibited calcium entry and subsequent NFAT activation.
- The authors measured intracellular calcium levels using Fura-2-AM and confirmed that removing extracellular ATP impaired calcium influx, suggesting that ATP acts through P2X7 receptors to facilitate this process.
IL-2 Production and NFAT Activation:
- Addition of exogenous ATP (at concentrations similar to endogenously released ATP) enhanced IL-2 mRNA expression by 47-fold compared to control cells.
- The study showed that blocking P2X7 receptors with selective inhibitors (o-ATP, suramin, and NF023) significantly reduced IL-2 production in human peripheral blood mononuclear cells (PBMCs).
- Using a NFAT-luciferase reporter assay, they demonstrated that overexpression of wild-type P2X7 receptors doubled NFAT activity in response to TCR/CD28 stimulation, while a nonfunctional mutant P2X7 receptor (T1729A) suppressed NFAT activation.
P2X7 Receptor Expression:
- Real-time PCR and immunohistochemistry confirmed that Jurkat T cells and human CD4+ T cells express P2X7 receptors, with expression levels increasing after T-cell stimulation.
- P2X7 receptor silencing (via siRNA) reduced receptor mRNA levels by 90%, resulting in impaired calcium signaling and IL-2 transcription. This highlights the critical role of P2X7 in T-cell activation.
Mouse Model Comparisons:
- BALB/c mice, which have fully functional P2X7 receptors, exhibited robust IL-2 production in response to phytohemagglutinin (PHA) stimulation.
- C57BL/6 mice, with a natural mutation impairing P2X7 function, showed significantly reduced IL-2 production, further supporting the importance of P2X7 receptors in T-cell activation.
ATP Release Mechanisms:
- The study suggested that ATP is released through maxi-anion channels, as the channel blocker GdCl3 reduced ATP release and IL-2 production, though it did not completely abolish these effects, indicating that other mechanisms (such as pannexin-1 channels) might also be involved.
Proliferation vs. Apoptosis:
- Jurkat cells are more resistant to ATP-induced apoptosis (even at high ATP concentrations, up to 3 mM) compared to primary CD4+ T cells. This difference is attributed to the lower expression of P2X7 receptors in Jurkat cells.
- In primary CD4+ T cells, P2X7 receptor overexpression is linked to apoptosis at ATP concentrations >100 μM, while lower ATP levels promote T-cell activation and proliferation.

Implications:

P2X7 receptors are essential for linking ATP release to downstream signaling pathways critical for T-cell activation. The findings indicate that ATP acts in an autocrine loop to reinforce TCR-mediated activation, leading to calcium influx, NFAT activation, and IL-2 production.
The study suggests that modulating ATP release or P2X7 receptor function could be a potential therapeutic strategy for immune modulation, particularly in disorders involving T-cell dysfunction or overactivation (e.g., autoimmunity or chronic inflammatory conditions).

Figures with Additional Details:

Figure 1: Demonstrates the ATP release near the T-cell surface upon stimulation, using fluorescent microscopy and HPLC to measure ATP levels.
Figure 2: Shows the role of ATP in IL-2 mRNA expression and the effects of the maxi-anion channel blocker GdCl3 in reducing ATP release.
Figure 3: Illustrates how ATP scavenging inhibits calcium signaling and IL-2 expression, confirming the necessity of extracellular ATP for T-cell activation.
Figure 5: Depicts P2X7 receptor expression in Jurkat and human CD4+ T cells and highlights the enhancement of NFAT activation with wild-type P2X7 overexpression.
Figure 6: Demonstrates the effectiveness of P2X7 receptor silencing by siRNA and its impact on calcium signaling and IL-2 transcription.

Conclusion:

This detailed study provides strong evidence that ATP release and P2X7 receptor activation play a central role in regulating T-cell activation, from calcium influx to NFAT-mediated IL-2 production. The autocrine feedback loop involving ATP and P2X7 receptors represents a critical checkpoint in T-cell signaling, suggesting that targeting this pathway could offer new approaches for immunotherapy.

Key Signaling Pathways : Autocrine Regulation of T-cell Activation by ATP Release and P2X7 Receptors

T-cell Receptor (TCR) Activation Leads to ATP Release:

TCR Activation \to ATP Release from T-cells

Extracellular ATP Acts Autocrinely on P2X7 Receptors:

Extracellular ATP + P2X7 Receptor \to P2X7 Receptor Activation

P2X7 Receptor Activation Induces Ca²⁺ Influx:

P2X7 Activation \to↑ {Ca}^{2 +} Influx \to↑ [{Ca}^{2 +}]_{i}

Increased Cytosolic Ca²⁺ Leads to NFAT Activation:

↑ [{Ca}^{2 +}]_{i} \to Calcineurin Activation \to Dephosphorylation of NFAT \to NFAT Translocation to Nucleus

NFAT Activation Promotes IL-2 Gene Transcription:

NFAT in Nucleus \to IL-2 Gene Transcription \to IL-2 Production

IL-2 Production Enhances T-cell Proliferation:

IL-2 \to Binding to IL-2 Receptor \to T-cell Proliferation and Activation

ATP Scavengers and P2X7 Inhibitors Reduce Ca²⁺ Influx and IL-2 Production:

ATP Scavengers (Apyrase, Alkaline Phosphatase):

ATP Scavengers \to↓ Extracellular ATP \to↓ P2X7 Activation \to↓ [{Ca}^{2 +}]_{i} \to↓ IL-2 Production

P2X7 Receptor Inhibitors (o-ATP, Suramin, NF023):

P2X7 Inhibitors \to Blocked P2X7 Activation \to↓ [{Ca}^{2 +}]_{i} \to↓ NFAT Activation \to↓ IL-2 Production

siRNA Silencing of P2X7 Receptors Impairs T-cell Activation:

P2X7 siRNA \to↓ P2X7 Expression \to↓ {Ca}^{2 +} Influx \to↓ NFAT Activation \to↓ IL-2 Production

Overexpression of P2X7 Receptors Enhances NFAT Activation:

↑ P2X7 Expression \to↑ {Ca}^{2 +} Influx \to↑ NFAT Activation \to↑ IL-2 Production

Maxi-Anion Channels Contribute to ATP Release:

TCR Activation \to Opening of Maxi-Anion Channels \to ATP Release

Maxi-Anion Channel Blocker (GdCl₃) Reduces ATP Release:

{GdCl}_{3} \to Inhibition of Maxi-Anion Channels \to↓ ATP Release \to↓ P2X7 Activation

High ATP Concentrations Induce Apoptosis via P2X7:

High [ATP] (> 100 μ M) \to Overactivation of P2X7 \to Apoptosis in T-cells

Summary of the Autocrine Feedback Loop:

TCR/CD28 Activation \to ATP Release \to P2X7 Activation \to↑ [{Ca}^{2 +}]_{i} \to NFAT Activation \to IL-2 Production \to T-cell Proliferation