Dr. Spelsberg's laboratory is engaged in studies of the mechanism of action of steroid hormones and the growth factor, TGFß in human bone and breast cancer tissues.

In projects 1 (osteoblast biology) and 2 (breast cancer), the actions of estrogen and SERMs on human breast cancer cells as well as in normal and transformed human osteoblast cells (for osteoporosis research) are being investigated. The latter includes immortalized fetal human osteoblast cell lines that were developed by this laboratory. These studies involve the estrogen effects on early gene- and late gene-expression, including those of bone matrix proteins, growth factors, and cytokines. We are currently examining the role of another form (i.e., the beta isoform) of the estrogen receptor (ERa), as well as its ariant, ERßcx on breast cancer and bone cell functions and gene expression. We have compared the actions of ERa to those of ERß and ERßcx, as well as the interactions and antagonists that these receptors display when co-expressed in both osteoblasts and breast cancer cells. Current studies are examining the molecular interactions of ERa and ERß at the gene transcription level, using chromatin immunoprecipitation assays and deletion constructs. The TIEG gene (described below) and RBP-1 (Rb binding protein) are the ERß and ERa specific genes respectively.

In related studies, we are measuring the actions and interactions of steroid receptor co-regulators (SRCs) with the ERa and ERß both in cell cultures and in SRC knockout mice, as well as the differential interactions of the ER isoforms with different enhancer elements. We are also examining the role of the TGFß/Smad pathway, a target of E2 and SERMs, playing a role in breast cancer cell non-responsiveness to SERMs.

The third project addresses the actions and interactions of TGFß and estrogen on human osteoblast cells in culture. Our laboratory discovered a novel TGFß/estrogen inducible early gene (TIEG1) which is a member of the Krüppel-like, 3 zinc finger, family of transcription factors. TIEG1 was shown to be a Krüppel-like, nuclear, transcription factor, whose expression is rapidly induced upon TGFß treatment. A major goal is to identify functional roles for TIEG, including whether or not it mediates TGFß or estrogen actions in human osteoblasts, osteosarcomas, and other breast cancer cells. TIEG is a part of the TGFß signaling pathway, is a possible tumor suppressor gene and plays a role in cancer, especially with regard to TGFß-Smad pathway. In clinical studies with human tissues, TIEG shows a tissue and cell-type specificity. TIEG gene knockout mice have been generated to examine the developmental and biological functions of TIEG. These female TIEG null mice display bone loss (osteopenia)and more fragile bones compared to wild-type. The male TIEG null mice show no effect. The role of sex steroids in this gender specificity is underway.

Using yeast two hybrid systems, gene arrays, chromatin immunoprecipitation assays, viral expression vectors and si- and sh-RNAs, overexpressing cell lines, we are attempting to identify the signal pathway(s) and co-regulators which involve TIEG. To date, we have evidence that TIEG activates the TGFß-Smad 2, 3, 4 pathway by repressing Smad 7 gene transcription and by activating Smad 2 gene expression. The identity of the regulatory elements in the Smad 7 promoter which bind the TIEG protein is also underway. TIEG protein was recently shown to induce Runx2 gene expression and to interact with the Runx2 protein to regulate other genes. Further TIEG was shown to repress OPG and enhance RANKL expression, thus affecting osteoclastogenesis.

In a fourth project, we are also examining TIEG's role in the development of human cancers. The TIEG gene has been co-localized with the non-lymphoma leukemia gene on chromosome 8,q22.2, and is deleted or translocated in many of the transformed cells in these leukemia patients. We have found and reported that the expression of TIEG is abolished in breast epithelial cells as they become malignant. Subsequent studies using Real Time PCR has shown a predictive remarkable inverse correlation between TIEG mRNA levels and the malignant state of breast biopsies. TIEG mRNA levels have been shown to serve as powerful predictive marker for cancer versus normal cells/tissues and combining TIEG mRNA levels with select other Smad markers, serves as a powerful predictor of cancer patient progress. This project is being combined with Project 2 described above.

In a fifth project we are collaborating with a former Mayo cardiology fellow, Dr. Rajamannan, now at Northwestern's Feinberg Medical Center, on the role of lipids and bone formation/calcification of heart valves and the role of TGFß/TIEG as a cause of hypertrophic cardiomyopathy. The TIEG knockout male mice, but not female mice, display this disease.

Techniques used: The projects described above involve the measurement of growth actor/cytokine mRNA and protein levels using ELISA, RIA, as well as molecular biology techniques, e.g., Northern, Western, Southern, and Southwestern blotting, DNA gel shift assays, polymerase chain reaction, chromatin immunoprecipitation assays (ChIP) and double ChIP assays cloning, gene deletions and mutations, gene transfections, CAT assays, gene knockout and transgenic animals, yeast two hybrid systems, gene microarrays, adenoviral expression systems, silencing (si) RNAs, as well as biochemical techniques of immunohistochemistry, confocal microscopy, real time PCR, DNA/protein sequencing, HPLC, 2-dimensional gel electrophoresis and mass spectrometry.