Thottala Jayaraman

T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis.

Tumor Necrosis Factor α is a Key Modulator of Inflammation in Cerebral Aneurysms

OBJECTIVE: Although intracranial aneurysms (IAs) are a major public health problem in the United States, few etiological factors are known. Most aneurysms remain asymptomatic until they rupture, producing subarachnoid hemorrhage, one of the most severe forms of stroke. Despite the technical advances in endovascular and microsurgical treatment, these patients still have high mortality and morbidity rates. Hence, the biology of aneurysm formation and growth is of intense interest. The presence of T and B lymphocytes, as well as macrophages, in human IA tissues suggests a role for inflammation in IA pathogenesis. However, the types of cytokines that are involved and regulated during cerebral aneurysm formation and growth are not known. To study the underlying pathogenesis of IA, we analyzed the expression of cytokines that participate in proinflammatory and anti-inflammatory responses. METHODS: Polymerase chain reaction was used to assess relative messenger ribonucleic acid expression levels of cytokines and an apoptotic modulator, Fas-associated death domain protein. Western blot analysis was used to determine protein expression from these genes. RESULTS: We show that the proinflammatory cytokine, tumor necrosis factor α and its proapoptotic downstream target, Fas-associated death domain protein, are increased in human aneurysms. In contrast, interleukin 10, which is secreted predominantly by T helper 2 cells, was absent in aneurysms. Polymerase chain reaction-derived gene expression data were confirmed by Western blotting using specific antibodies. CONCLUSION: Increased tumor necrosis factor α and Fas-associated death domain protein may have deleterious primary and secondary effects on cerebral arteries by promoting inflammation and subsequent apoptosis in vascular and immune cells, thereby weakening vessel walls.

TNF-α-mediated inflammation in cerebral aneurysms: A potential link to growth and rupture

Intracranial aneurysm (IA) rupture is one of the leading causes of stroke in the United States and remains a major health concern today. Most aneurysms are asymptomatic with a minor percentage of rupture annually. Regardless, IA rupture has a devastatingly high mortality rate and does not have specific drugs that stabilize or prevent aneurysm rupture, though other preventive therapeutic options such as clipping and coiling of incidental aneurysms are available to clinicians. The lack of specific drugs to limit aneurysm growth and rupture is, in part, attributed to the limited knowledge on the biology of IA growth and rupture. Though inflammatory macrophages and lymphocytes infiltrate the aneurysm wall, a link between their presence and aneurysm growth with subsequent rupture is not completely understood. Given our published results that demonstrate that the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is highly expressed in human ruptured aneurysms, we hypothesize that pro-inflammatory cell types are the prime source of TNF-alpha that initiate damage to endothelium, smooth muscle cells (SMC) and internal elastic lamina (IEL). To gain insights into TNF-alpha expression in the aneurysm wall, we have examined the potential regulators of TNF-alpha and report that higher TNF-alpha expression correlates with increased expression of intracellular calcium release channels that regulate intracellular calcium (Ca2+), and Toll like receptors (TLR) that mediate innate immunity. Moreover, the reduction of tissue inhibitor of metalloproteinase-1 (TIMP-1) expression provides insights on why higher matrix metalloproteinase (MMP) activity is noted in ruptured IA. Because TNF-alpha is known to amplify several signaling pathways leading to inflammation, apoptosis and tissue degradation, we will review the potential role of TNF-alpha in IA formation, growth and rupture. Neutralizing TNF-alpha action in the aneurysm wall may have a beneficial effect in preventing aneurysm growth by reducing inflammation and arterial remodeling.

Inositol 1,4,5-trisphosphate receptor (type 1) phosphorylation and modulation by Cdc2

Calcium (Ca2+) release from the endoplasmic reticulum (ER) controls numerous cellular functions including proliferation, and is regulated in part by inositol 1,4,5‐trisphosphate receptors (IP3Rs). IP3Rs are ubiquitously expressed intracellular Ca2+‐release channels found in many cell types. Although IP3R‐mediated Ca2+ release has been implicated in cellular proliferation, the biochemical pathways that modulate intracellular Ca2+ release during cell cycle progression are not known. Sequence analysis of IP3R1 reveals the presence of two putative phosphorylation sites for cyclin‐dependent kinases (cdks). In the present study, we show that cdc2/CyB, a critical regulator of eukaryotic cell cycle progression, phosphorylates IP3R1 in vitro and in vivo at both Ser421 and Thr799 and that this phosphorylation increases IP3 binding. Taken together, these results indicate that IP3R1 may be a specific target for cdc2/CyB during cell cycle progression.

Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

Novel translocation (9;12)(q22;q24) in secondardy chondrosarcoma arising from hereditary multiple exostosis

We report a new translocation in a patient with a history of hereditary multiple exostosis (HME) who developed a recurrent grade I chondrosarcoma involving the sacrum and retroperitoneum. Karyotypic analysis of the tumor revealed a sole chromosome abnormality t(9;12)(q22;q24.3). To our knowledge, this translocation has not been previously identified in either chondrosarcoma, HME, or related tumor types. Our novel translocation may be related to the sarcomatous degeneration of the pre-existing exostosis.

Calcium-Dependent Signalling in Apoptosis

Organogenesis requires the death of specific cells at genetically determined time points during development. This type of cell death, known as programmed cell death or apoptosis, plays a major role in the normal development of most organs including the immune and central nervous systems. Kerr and coworkers first described the ultrastructural changes characteristic of dying cells (Kerr et al., 1972) and since then few fields have attracted as much attention as apoptosis. Apoptosis is a natural process, and its dysregulation is pathologic. For example, apoptosis is believed to contribute to neuronal loss during stroke or central nervous system trauma and in neurodegenerative disorders including Alzheimer’s and Parkinson’s disease. Conversely, lack of apoptosis is known to result in tumorigenesis. Cells are genetically programmed for self-repair following pathological insult. Under normal conditions when self-repair is unsuccessful a series of highly regulated suicide signaling pathways are initiated. Apoptosis is triggered by extrinsic or intrinsic inducers that activate pathways leading to DNA cleavage. Emerging paradigms for apoptotic signaling include: 1) the requirement for translocation of proteins from the cytosol to the nucleus; and 2) calcium (Ca2+)-dependent activation of phosphatases required for this translocation. Cytosolic Ca2+ is a second messenger that regulates signaling required for cellular activation, proliferation, differentiation and cell death. Cytosolic Ca2+ is rigidly maintained at ~100 nM in resting cells and its elevation to levels between 500 and 1000 nM activates numerous signaling pathways in all types of cells. Levels above 1000 nM are generally toxic. Ca2+ channels in the plasma membrane (both voltage- and ligand-gated), and ligand-gated Ca2+-release channels in the endoplasmic reticulum are the most important sources of cytosolic Ca2+. Conversely, Ca2+-ATPases on endoplasmic reticulum and the plasma membrane are the most important pathways for removal of cytosolic Ca2+.