Abdallah Badou

Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9†

Apoptosis of hepatocytes results in the development of liver fibrosis, but the molecular signals mediating this are poorly understood. Degradation and modification of nuclear DNA is a central feature of apoptosis, and DNA from apoptotic mammalian cells is known to activate immune cells via Toll‐like receptor 9 (TLR9). We tested if DNA from apoptotic hepatocytes can induce hepatic stellate cell (HSC) differentiation. Our data show that apoptotic hepatocyte DNA and cytidine‐phosphate‐guanosine oligonucleotides induced up‐regulation of transforming growth factor β1 and collagen 1 messenger RNA both in the human HSC line LX‐2 and in primary mouse HSCs. These effects were opposed by TLR9 antagonists. We have recently shown that adenosine inhibits HSC chemotaxis, and we now show that apoptotic hepatocyte DNA also inhibits platelet‐derived growth factor (PDGF)‐mediated HSC chemotaxis. Inhibition of HSC chemotaxis by PDGF was blocked by TLR9 antagonists, and was absent in primary HSCs from mice deficient in TLR9 or the TLR adaptor molecule MyD88. Stimulation of TLR9 on HSCs blocked signaling by the PDGF signaling molecule inositol 1,4,5‐triphosphate and reduced PDGF‐mediated increase in cytosolic Ca2+. Conclusion: DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by (1) providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and (2) inducing a stationary phenotype‐associated up‐regulation of collagen production. (HEPATOLOGY 2007.)

Critical role for the β regulatory subunits of Cav channels in T lymphocyte function

Calcium ion is a universal signaling intermediate, which is known to control various biological processes. In excitable cells, voltage-gated calcium channels (Cav) are the major route of calcium entry and regulate multiple functions such as contraction, neurotransmitter release, and gene transcription. Here we show that T lymphocytes, which are nonexcitable cells, express both regulatory β and pore-forming Cav1 α1 subunits of Cav channels, and we provide genetic evidence for a critical role of the Cav β3 and Cav β4 regulatory subunits in T lymphocyte function. Cav β-deficient T lymphocytes fail to acquire normal functions, and they display impairment in the T cell receptor-mediated calcium response, nuclear factor of activated T cells activation, and cytokine production. In addition, unlike in excitable cells, our data suggest a minimal physiological role for depolarization in Cav channel opening in T cells. T cell receptor stimulation induces only a small depolarization of T cells, and artificial depolarization of T cells using KCl does not lead to calcium entry. These observations suggest that the Cav channels expressed by T cells have adopted novel regulation/gating mechanisms.

A Scaffold Protein, AHNAK1, Is Required for Calcium Signaling during T Cell Activation

Engagement of the T cell antigen receptor (TCR) during antigen presentation initiates a coordinated action of a large number of signaling proteins and ion channels. AHNAK1 is a scaffold protein, highly expressed by CD4+ T cells, and is a critical component for calcium signaling. We showed that AHNAK1-deficient mice were highly susceptible to Leishmania major infection. AHNAK1-deficient CD4+ T cells responded poorly to TCR stimulation in vitro with low proliferation and low Interleukin-2 production. Furthermore, AHNAK1 deficiency resulted in a reduced calcium influx upon TCR crosslinking and subsequent poor activation of the transcription factor NFAT. AHNAK1 was required for plasma membrane expression of L-type calcium channels alpha 1S (Cav1.1), probably through its interaction with the beta regulatory subunit. Thus, AHNAK1 plays an essential role in T cell Ca2+ signaling through Cav1 channels, triggered via TCR activation; therefore, AHNAK1 is a potential target for therapeutic intervention.

Defective survival of naive CD8+ T lymphocytes in the absence of the |[beta]|3 regulatory subunit of voltage-gated calcium channels

The survival of T lymphocytes requires sustained, Ca2+ influx–dependent gene expression. The molecular mechanism that governs sustained Ca2+ influx in naive T lymphocytes is unknown. Here we report an essential role for the β3 regulatory subunit of voltage-gated calcium (Cav) channels in the maintenance of naive CD8+ T cells. Deficiency in β3 resulted in a profound survival defect of CD8+ T cells. This defect correlated with depletion of the pore-forming subunit Cav1.4 and attenuation of T cell antigen receptor (TCR)-mediated global Ca2+ entry in CD8+ T cells. Cav1.4 and β3 associated with T cell signaling machinery and Cav1.4 localized in lipid rafts. Our data demonstrate a mechanism by which Ca2+ entry is controlled by a Cav1.4-β3 channel complex in T cells.

Emerging Roles of L-Type Voltage-Gated and Other Calcium Channels in T Lymphocytes

In T lymphocytes, calcium ion controls a variety of biological processes including development, survival, proliferation, and effector functions. These distinct and specific roles are regulated by different calcium signals, which are generated by various plasma membrane calcium channels. The repertoire of calcium-conducting proteins in T lymphocytes includes store-operated CRAC channels, transient receptor potential channels, P2X channels, and L-type voltage-gated calcium (Cav1) channels. In this paper, we will focus mainly on the role of the Cav1 channels found expressed by T lymphocytes, where these channels appear to operate in a T cell receptor stimulation-dependent and voltage sensor independent manner. We will review their expression profile at various differentiation stages of CD4 and CD8 T lymphocytes. Then, we will present crucial genetic evidence in favor of a role of these Cav1 channels and related regulatory proteins in both CD4 and CD8 T cell functions such as proliferation, survival, cytokine production, and cytolysis. Finally, we will provide evidence and speculate on how these voltage-gated channels might function in the T lymphocyte, a non-excitable cell.

Requirement for AHNAK1-mediated calcium signaling during T lymphocyte cytolysis

Cytolytic CD8+ T cells (CTLs) kill virally infected cells, tumor cells, or other potentially autoreactive T cells in a calcium-dependent manner. To date, the molecular mechanism that leads to calcium intake during CTL differentiation and function has remained unresolved. We demonstrate that desmoyokin (AHNAK1) is expressed in mature CTLs, but not in naive CD8+ T cells, and is critical for calcium entry required for their proper function during immune response. We show that mature AHNAK1-deficient CTLs exhibit reduced Cav1.1 α1 subunit expression (also referred to as L-type calcium channels or α1S pore-forming subunits), which recently were suggested to play a role in calcium entry into CD4+ T cells. AHNAK1-deficient CTLs show marked reduction in granzyme-B production, cytolytic activity, and IFN-γ secretion after T cell receptor stimulation. Our results demonstrate an AHNAK1-dependent mechanism controlling calcium entry during CTL effector function.

Activation of CD4 T cells by Raf-independent effectors of Ras

Small GTPase Ras is capable of mediating activation in T lymphocytes by using Raf kinase-dependent signaling pathway. Other effectors of Ras exist, however, suggesting that targets of Ras alternative to Raf may also contribute to T cell functions. Here we demonstrate that RasV12G37 mutant that fails to bind Raf, potently increases intracellular calcium concentration and cytokine production in primary antigen-stimulated T cells. From three known effectors which retain the ability to interact with RasV12G37, overexpression of phospholipase C ɛ but not that of RIN1 or Ral guanine nucleotide exchange factors enhanced cytokine and nuclear factor-activated T cell reporter T cell responses. Hence T cell activation can be critically regulated by the Ras effector pathway independent from Raf that can be mimicked by phospholipase C ɛ.

T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.