Janet L. Stein

Phosphorylation at serine 208 of the 1α,25-dihydroxy Vitamin D3 receptor modulates the interaction with transcriptional coactivators

Upon ligand binding the 1alpha,25-dihydroxy Vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1alpha,25-dihydroxy Vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRalpha. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.

Integrin alphavbeta6 promotes an osteolytic program in cancer cells by upregulating MMP2

The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the αvβ6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency associated peptide-TGFβ1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the αvβ6 integrin promotes this type of metastasis. We show for the first time that αvβ6 selectively induces matrix metalloproteinase 2, MMP2, in vitro in multiple prostate cancer cells, and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of αvβ6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of PTHrP, known to induce osteoclastogenesis, were also observed in αvβ6 expressing cells. However, using MMP2 shRNA, we demonstrate that the αvβ6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not to changes in tumor growth rate. Another related αv-containing integrin, αvβ5, fails to show similar responses, underscoring the significance of αvβ6 activity. Overall, these mechanistic studies establish that expression of a single integrin, αvβ6, contributes to the cancer cell -mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy of metastatic bone disease.The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the αvβ6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency-associated peptide-TGF-β1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the αvβ6 integrin promotes this type of metastasis. We show for the first time that αvβ6 selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of αvβ6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of parathyroid hormone-related protein (PTHrP), known to induce osteoclastogenesis, were also observed in αvβ6-expressing cells. However, by using MMP2 short hairpin RNA, we demonstrate that the αvβ6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not due to changes in tumor growth rate. Another related αv-containing integrin, αvβ5, fails to show similar responses, underscoring the significance of αvβ6 activity. Overall, these mechanistic studies establish that expression of a single integrin, αvβ6, contributes to the cancer cell-mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease.

Cell Cycle Control of Transcription at the G1/S Phase Transition

Competency for proliferation and cell cycle progression are functionally linked to the activities of regulatory factors that modulate the cell division cycle in response to the multi-directional signals of cell type specific signaling cascades. The parameters that mediate growth control are complex and interdependent, and whether cells grow or cease division reflects the integration of a broad spectrum of positive and negative growth regulatory signals that operate within distinct biological contexts. Equally important from a clinical perspective, fidelity of cell cycle regulatory mechanisms is compromised in transformed and tumor cells and in nonmalignant disorders, and reflects deregulation of cell growth or abrogation of cell death.

Epigenetic Changes Associated with Chromosomal Translocation in Leukemia

Soraya Gutierrez1, Amjad Javed2, Janet Stein3, Gary Stein3, Sandra Nicovani4, Valentina Fernandez1, Ricardo Alarcon1, Marcela Stuardo1, Milka Martinez1, Marcela Hinojosa1 and Boris Rebolledo-Jaramillo1 1Universidad de Concepcion, Departamento de Bioquimica y Biologia Molecular, 2University of Alabama Department of Oral and Maxillofacial Surgery 3University of Massachusetts 4Universidad Santo Tomas; 1,4Chile, 2.3USA

Erratum: Bipartite structure of the proximal promoter of a human H4 histone gene (Journal of Cellular Biochemistry (1995) 58 (372-379))

The proximal promoter of the human H4 histone gene FO108 contains two regions of in vivo protein‐DNA interaction, Sites I and II. electrophoretic, mobility shift assays using a radiolabeled DNA probe revealed that several proteins present in HeLa cell nuclear extracts bound specifically to Site 1 (nt‐125 to nt‐86). The most prominent complex, designated HiNF‐C, and a complex of greater mobility, HiNF‐C′, using were specifically competed by an Sp1 consensus oligonucleotide. Fractionation of HiNF‐C using wheat germ agglutinin affinity chromatography suggested that, like Sp1, HiNF‐C contains N‐acetylglucosamine moieties. Two minor complexes of even greater mobility, designated HiNF‐E and F, were competed by ATF consensus oligonucleotides. A DNA probe carrying a site‐specific mutation in the distal portion of Site I failed to bind HiNF‐E, indicating that this protein associated specifically to this region. UV cross‐linking analysis showed that several proteins of different molecular wieghts interact specifically with Site I. These data indicate that Site I possesses as bipartite structure and that multiple proteins present in HeLa cell nuclear extracts interact specifically with Site I sequences.