Jaroslaw Jozwiak

Possible mechanisms of disease development in tuberous sclerosis

The two-hit hypothesis presented by Knudson in 1971 explains the development of tumours deficient in anti-oncogenes. Hamartomas in patients with tuberous sclerosis usually fit into this model, the first hit is a congenital lesion of either of the tuberous sclerosis genes (TSC1 or TSC2), and the second hit is loss of heterozygosity of this gene. Although this mechanism is true for most tumours associated with tuberous sclerosis, only 30-60% of brain and cardiac tumours show loss of heterozygosity--the remaining tumours develop despite the presence of an intact allele. Tumours in which loss of heterozygosity is rare, such as subependymal giant-cell astrocytoma, might all share a common feature that mimics loss of heterozygosity either by inactivation of the TSC complex or by direct activation of mammalian target of rapamycin (mTOR) or its downstream targets. Because phosphorylation of the TSC complex can inactivate it, expression and activation patterns of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK), two potent protein kinases that are activators of the mTOR pathway, have been implicated. AKT activation is detected only in few samples, whereas ERK is hyperactive in all subependymal giant-cell astrocytomas. We postulate that ERK activation consistently detected in different tuberous-sclerosis-associated tumours is a molecular trigger for the development of these neoplasms.

Hamartin and tuberin: Working together for tumour suppression

TSC1 and TSC2 are two recently identified tumour suppressor genes encoding hamartin and tuberin, respectively, and involved in pathogenesis of tuberous sclerosis, neurological disorder connected with the development of hamartomas in numerous organ systems, including the brain, kidneys, heart and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase‐activating (GAP) activity towards a small G protein, Ras homologue enriched in brain (Rheb). Inhibition of Rheb is important for the regulation of mTOR pathway, while mutation of hamartin or tuberin results in uncontrolled cell cycle progression. Tuberin, possessing the Rheb‐GAP domain, is phosphorylated by several kinases that confer the signals of growth factor stimulation or low cellular energy levels. Such a modification of tuberin influences its activity within the complex with hamartin and positively or negatively modulates mTOR‐regulated protein translation and cellular proliferation. Current article describes biochemical properties of hamartin and tuberin, their known regulatory phosphorylation sites and binding partners.

Positive and negative regulation of TSC2 activity and its effects on downstream effectors of the mTOR pathway

Tuberous sclerosis is an autosomal-dominant disorder caused by the mutation of one of the two tumor suppressor genes: TSC1 or TSC2, encoding protein products, hamartin, and tuberin, respectively. Both proteins form intracellular complexes exerting inhibitory activity on mammalian target of rapamycin (mTOR) kinase. It has been demonstrated that signal transduction from tuberin to mTOR is mediated by a G protein, Ras homologue enriched in brain (Rheb). In normal cells, tuberin having GTPase-activating protein properties toward Rheb controls signals of nutrient depletion, hypoxia, or stress, not allowing activation of mTOR and subsequent protein translation and cell proliferation. However, when environmental conditions change, tuberin is phosphorylated and it forms a complex with hamartin is degraded, and downstream targets of mTOR, S6K, and eEF2K, can be activated. In this review, we summarize very recent information contributing to our knowledge of TSC2 regulation by four cellular signaling pathways: PI3K/Akt, Ras/MAPK, LKB1/AMPK, and REDD1.

Giant Cells: Contradiction to Two-Hit Model of Tuber Formation?

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by the formation of hamartomatous lesions in many organs, including brain, heart or kidneys. It has been found that TSC is caused by the mutation in one of the two tumor suppressor genes: TSC1 or TSC2, encoding hamartin and tuberin, respectively. According to Knudson’s two-hit model of tumorigenesis, second-hit mutation and resulting loss of heterozygosity (LOH) of a tumor suppressor gene is necessary for tumor formation. In fact, LOH is commonly found in several types of hamartomas formed in the process of tuberous sclerosis, but, interestingly, not in brain lesions, containing characteristic giant cells. In this paper, we review literature covering origination of giant cells and present several hypotheses explaining why in spite of the presence of hamartin and tuberin, brain lesions form in TSC patients.

Hamartin and tuberin modulate gene transcription via β-catenin

SummaryTuberous sclerosis, neurological genetic disorder characterized by the formation of benign tumors or hamartomas in multiple organ systems, is recently getting much attention. Numerous papers describe still-not-fully-explained pathogenesis of the disease. Studies on tuberous sclerosis allowed identification of two tumor suppressor genes, TSC1 and TSC2, encoding proteins implicated in the disease: hamartin and tuberin, respectively. The importance of these proteins is confirmed by their ubiquitous character and by the fact that TSC1/TSC2 complex is involved in the regulation of the activity of mTOR, a master controller of protein translation. Thus, the meaning of hamartin and tuberin goes far beyond tuberous sclerosis. As far as the influence of the TSC1/TSC2 complex on protein translation is well described in numerous reviews, little attention is drawn to the recently discovered role of the TSC1/TSC2 complex in gene transcription via the WNT signaling pathway. The present paper focuses on recent developments documenting the role of hamartin and tuberin in the WNT pathway.

Brain tumor formation in tuberous sclerosis depends on Erk activation.

Tuberous sclerosis (TS) is an autosomal dominant disease associated with the formation of usually benign tumors or hamartomas. The disease is connected with upregulation of mammalian target of rapamycin, central regulator of protein translation, which is usually regarded to be activated by Akt kinase. Here, we show for the first time that in all four brain lesions and one angiomyolipoma from TS patients both extracellular signal-regulated kinase (Erk) and p90 ribosomal S6 kinase 1 activation as well as Erk-dependent phosphorylation of p70 ribosomal S6 kinase 1 are markedly elevated whereas Akt, participating in the classical pathway of mammalian target of rapamycin activation is not always activated. Erk activation is also present in TS-derived cell lines. Importantly, Erk inhibition leads to the decrease of proliferation potential of such lines. These results show that Erk is specifically implicated in the pathogenesis of hamartomas.

Upregulation of the WNT pathway in tuberous sclerosis-associated subependymal giant cell astrocytomas

Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10-15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that beta-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by beta-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs.

Transplants of rat chondrocytes evoke strong humoral response against chondrocyte-associated antigen in rabbits.

Rat chondrocytes transplanted intramuscularly in rabbits produced cartilage. In 1-day-old transplants chondrocytes remained viable. After 1 week peripheral chondrocytes of the transplant were dead and the cartilage was surrounded and resorbed by macrophages. In 2-week-old transplants cartilage deteriorated and was invaded by fibroblast-like cells and macrophages. Sera of rabbits that received two or three consecutive transplants of rat chondrocytes with 2-week intervals contained high titer of antichondrocyte cytotoxic antibodies. A part of the cytotoxic activity could be removed by absorption with rat splenocytes. Western blot analysis of lysates from fresh or 24-h cultured chondrocytes with absorbed sera detected antigen with Mr of ~74 and ~23 kDa. Only the latter remained after reduction in 2-mercaptoethanol. In lysates of fibroblasts and endotheliocytes the 23-kDa antigen was not found but the serum reacted with Mr 39-kDa antigen. In lysates of thymocytes a weak band corresponding to Mr of 35 kDa was present. Serum from rabbits receiving transplants of living chondrocytes followed by chondrocytes suspended in complete Freunds adjuvant contained antibodies directed against components of crude collagenase used for cell isolation. Such antibodies could not be detected in sera of rabbits receiving transplants of living chondrocytes only. Molecular weight of detected antigen differs from that of collagen type II, core of aggrecan, link proteins, and several other macromolecules of cartilage matrix. It could represent either a component of chondrocyte membrane or a membrane-bound substance resistant to enzymes used for isolation. Availability of antibodies against presumably chondrocyte-specific antigen produced during transplant rejection may help to characterize it more precisely and to ascertain whether its presence may influence results of autogenous chondrocyte transplants in humans.

Similarity of Balloon Cells in Focal Cortical Dysplasia to Giant Cells in Tuberous Sclerosis

To the Editor: In a very recent article in Epilepsia (2005; 46:1716– 1723), Ying et al. studied balloon cells from focal cortical dysplasia (FCD), coming to the conclusion that the cells form a heterogeneous group with differential expression of glial/astrocytic markers [microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP)] and several other tissue-specific proteins. Such a result resembles our and other authors’ conclusions from immunohistochemical and ultrastructural research on giant cells in tuberous sclerosis (TS). We found characteristic cells present in all three types of TS-associated lesions: subependymal giant cell astrocytoma (SEGA), cortical tubers, and subependymal nodules (SENs) share a similar profile of protein expression and are a group of undifferentiated cells that, depending on individual determination, can show neural or glial features (Jozwiak et al. Immunohistochemical and microscopic studies on giant cells in tuberous sclerosis. Histol Histopathol 2005;20:1321– 1326). Thus we recently evaluated the presence of two TSassociated proteins, hamartin and tuberin, in FCD type I (in which balloon cells are not found) and IIB (with balloon cells). Hamartin and tuberin are products of tumorsuppressor genes TSC1 and TSC2, inevitable for proper development of tissues and lethal if both alleles are subject to germ-line mutation. Inactivating mutation of TSC1 or TSC2, found in TS, leads to upregulation of mTOR (mammalian target of rapamycin), the kinase being a central regulator of protein transcription and subsequent uncontrolled cell proliferation. As a result, the clinical manifestation of TS is associated with tumor development in the brain, heart, kidneys, lungs, liver, and/or skin. Based on similarities between balloon and giant cells, our hypothesis was that FCD may be a form of focal TS, limited to selected regions of the brain tissue. According to our expectations, in an immunohistochemical study, we found loss of tuberin and hamartin expression, as well as strong immunoreactivity for mTOR in FCD type IIB. Specific kinases implicated in TS progression (pS6K1, pAkt, and pErk) also were hyperactivated in the same samples. Cortical balloon cells in these samples were NSE positive, whereas most subcortical giant cells were labeled with GFAP. In FCD type I and normal brain tissue, tuberin and hamartin were detected, but immunoreactivity for mTOR, the active form of S6K1, Akt, and Erk was subtle. Nestin expression in all samples was weak or absent. These findings suggest activation of the same pathways underlying both FCD IIB and TS and argue for recognition of the similarities in both pathologies and for considering FCD IIB as a focal form of TS. Such a conclusion could also potentially have a practical outcome: current research with the application of rapamycin as an effective drug limiting activity of mTOR kinase are under way and, in animal models of TS, show effectiveness of this therapy. Nevertheless, further elucidation of accompanying mutiations that probably predispose patients to FCD must be performed.

Fibroblasts from normal skin of a tuberous sclerosis patient show upregulation of mTOR pathway.

Skin lesions are one of the characteristic features in tuberous sclerosis (TS), a neurocutaneous disorder caused by mutation of 1 of 2 tumor suppressor genes, encoding hamartin and tuberin. So far, however, studies on skin abnormalities present in TS patients are very few and do not contribute to the knowledge of the disease. In our current work, we cultured fibroblasts from healthy skin of a TS patient and evaluated upregulation of pathways found to be implicated in progression of TS tumors. We found that even healthy skin fibroblasts show upregulation of S6 ribosomal protein.