Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that shares clinical similarities with BHD. TSC results from germline mutations in the Tsc1 or Tsc2 gene, affecting multiple organs, including the kidney and lung. In the kidney, lesions such as multiple renal cysts and renal cell carcinoma can occur. In the lung, patients can develop multifocal micronodular pneumocyte hyperplasia and LAM. TSC proteins are negative regulators of the mTORC1 pathway. The mechanisms of organ-specific manifestations in TSC remain unknown, and the impact of TSC on mesenchymal lineage cells has not yet been addressed. In a recent study, Ren et al. (2016) deleted Tsc2 specifically in mesoderm-derived mesenchymal cells in a variety of organs in mice. Inactivation of Tsc2 in mesoderm-derived cells caused impaired body growth, premature death, increased cell proliferation in the kidneys but reduced cell proliferation in the lungs, renal cysts, and deficient lung alveolarization.
Conventional homozygous Tsc1 or Tsc2 knockout mice are embryonic lethal, and heterozygous knockout mice primarily develop epithelial renal lesions and tumours (Kobayashi et al., 1999; Onda et al.,1999; Kwiatkowski et al., 2002) in contrast to human TSC, in which lesions of the kidney and lung have mesenchymal origin. Ren et al. investigated the function of Tsc2 specifically in mesoderm-derived cells by crossing a mesoderm-specific Cre-expressing mouse line Dermo1-Cre with floxed-Tsc2 mice to generate mesenchyme-specific Tsc2 conditional knockout mice (Tsc2 CKO). Tsc2 CKO mice showed clearly reduced body growth compared to their littermate controls. The homozygous Tsc2 CKO mice had a median survival of 20 days. At day 14 postnatal Tsc2 CKO mice, showed an abnormal kidney and lung phenotype.
Using fluorescence microscopy, the authors found that Dermo1-Cre–driven gene deletion occurred specifically in kidney mesenchymal cells, and not in endothelial nor tubular epithelial cells. Reduction of Tsc2 protein and activation of the mTORC1 pathway was confirmed by Western blotting. Tsc2 CKO mice developed severe polycystic kidney disease within 3 weeks after birth, resulting in abnormal kidney structure. Multiple foci of tubular epithelial hyperplasia and adenomatous changes adjacent to the cysts were seen in the kidney after 2 weeks. Overall cell proliferation in Tsc2 CKO kidneys was increased at day 7 after birth before obvious morphological changes occurred. Some of the tubular epithelial cells were positively stained with a mesenchymal cell marker in the Tsc2 CKO kidney, with significant reduction of epithelial cell marker expression, suggesting an epithelial-mesenchymal transition due to Tsc2 inactivation. In addition, increased apoptosis, detected by active caspase 3, was seen in Tsc2 CKO kidney sections. Only minor increase of serum cystatin C was detected in the Tsc2 CKO mice, suggesting that kidney function is not severely impaired. These results suggest that Tsc2 in renal mesenchymal cells is crucial for maintaining normal structural homeostasis after development but not for regulating kidney morphogenesis per se and that some of the epithelial cystic lesions in TSC may arise through mesenchymal loss of TSC2 function, supporting a new model for the cyst pathogenesis in TSC.
In the lung, the authors confirmed that most lung mesenchymal cells were targeted by the Dermo1-Cre, unlike endothelial or epithelial cells. As in the kidney, deletion of Tsc2 in the lung was validated by reduced Tsc2 protein expression and increased mTOR pathway activity. In the Tsc2 CKO mouse lung, growth of alveolar septa was markedly decreased, which resulted in larger alveolar spaces showing that lung mesenchymal Tsc2 has an important role in promoting lung alveolarization during postnatal development. In contrast to the kidney, a significant reduction of cell proliferation was detected at day 7 in the Tsc2 CKO mouse lungs. By examining the alveolar septa structure, the authors found that α-smooth muscle actin–positive myofibroblasts in Tsc2 CKO lung were significantly reduced in number. This suggests that reduced lung alveolarization in Tsc2 CKO mice is caused both by altered cell proliferation and myofibroblast differentiation. Whether defective alveolarization in these Tsc2 CKO mice contributes to pulmonary cystic lesions in adult remains unknown, because of the mice’s shortened life span: further investigation of the link between lung mesenchymal Tsc2 inactivation and LAM-like pathology is needed.
To summarize, mutation in Tsc2 and subsequent mTORC1 activation results in different cellular responses depending on cell lineages, organs, and stages of development and homeostasis. The authors reported the novel finding that homozygous Tsc2 CKO mice are viable but have reduced body growth, progressive polycystic kidney disease, and defective lung alveolarization.
Given the clinical similarities between BHD and TSC, and since FLCN and TSC are both linked to the mTOR pathway, the study of BHD will certainly benefit from TSC research and from comparisons between these two syndromes.
- Ren S, Luo Y, Chen H, Warburton D, Lam HC, Wang L, Chen P, Henske EP, & Shi W (2016). Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization. The American journal of pathology PMID: 27768862