Kerstin Tiedemann

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Interactions of Fibrillin-1 with Heparin/Heparan Sulfate, Implications for Microfibrillar Assembly

Fibrillin-1 is a major constituent of the 10–12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg45–Thr450) and C-terminal (Asp1528–Arg2731) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp1028–Thr1486) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by β-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Fibrillin-1 Interactions with Fibulins Depend on the First Hybrid Domain and Provide an Adaptor Function to Tropoelastin

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.

Fibrillins: From Biogenesis of Microfibrils to Signaling Functions

Fibrillins are large proteins that form extracellular microfibril suprastructures ubiquitously found in elastic and nonelastic tissues. Mutations in fibrillin-1 and -2 lead to a number of heritable connective tissue disorders generally termed fibrillinopathies. Clinical symptoms in fibrillinopathies manifest in the skeletal, ocular, and cardiovascular systems and highlight the importance of fibrillins in development and homeostasis of tissues and organs, including blood vessels, bone, and eye. Microfibrils appear to have dual roles in (1) conferring mechanical stability and limited elasticity to tissues, and (2) modulating the activity of growth factors of the transforming growth factor beta (TGF-beta) superfamily. This chapters focus is on the biogenesis of microfibrils, developmental expression patterns of fibrillins, signaling functions of microfibrils, and mouse models deficient in fibrillins.

Biosynthesis of Dermatan Sulfate CHONDROITIN-GLUCURONATE C5-EPIMERASE IS IDENTICAL TO SART2

We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts d-glucuronic acid to l-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an ∼43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565–2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.

CCN3 Impairs Osteoblast and Stimulates Osteoclast Differentiation to Favor Breast Cancer Metastasis to Bone

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patients quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Osteoclast precursors acquire sensitivity to breast cancer derived factors early in differentiation

The development of osteolytic breast cancer bone metastases relies on the ability of tumor cells to stimulate the formation of bone-resorbing osteoclasts. We have studied the effects of soluble factors produced by MDA-MB-231 breast carcinoma cells on osteoclast formation from human monocytic precursors and RAW 264.7 monocytic cells. Although factors produced by breast cancer cells were ineffective in inducing osteoclast formation from monocytes, priming with RANKL for 1-3 days dramatically increased receptiveness of osteoclast precursors to cancer-derived factors, which enhanced osteoclast formation 2-3 fold in the absence of supporting cell types. Osteoclasts formed by exposure to cancer factors expressed proteases critical for bone resorption, cathepsin K and matrix metalloproteinase 9, and were capable of resorbing calcified matrices. Expression of key osteoclastogenic transcription factor NFATc1 in osteoclast precursors was dramatically increased by short treatment with RANKL. NFATc1 was localized in the nuclei of primed osteoclast precursors when RANKL was present; however removal of RANKL led to rapid nuclear export of NFATc1. Cancer-derived factors were able to substitute for RANKL in supporting nuclear localization of NFATc1. Using neutralizing antibodies against TGFbeta, and a kinase inhibitor targeting the TGFbeta type I receptor, we identified TGFbeta as a permissive factor, required for the effects of breast cancer cells on NFATc1 nuclear accumulation and osteoclast formation. Our data suggest that, during differentiation, osteoclast precursors acquire the competency to respond to factors secreted by breast cancer cells, which may serve to promote tumor growth at skeletal sites undergoing active bone turnover.

Breast Cancer-derived Factors Stimulate Osteoclastogenesis through the Ca2+/Protein Kinase C and Transforming Growth Factor-β/MAPK Signaling Pathways

Breast cancer commonly metastasizes to bone where its growth depends on the action of bone-resorbing osteoclasts. We have previously shown that breast cancer cells secrete factors able to directly stimulate osteoclastogenesis from receptor activator of nuclear factor κB ligand (RANKL)-primed precursors and that transforming growth factor-β (TGFβ) plays a permissive role in this process. Now, we evaluate the signaling events triggered in osteoclast precursors by soluble factors produced by MDA-MB-231 human breast carcinoma cells. In mouse bone marrow cultures and RAW 264.7 murine monocytic cells, MDA-MB-231-derived factors increased osteoclast number, size, and nucleation. These factors failed to induce Smad2 phosphorylation, and short interfering RNAs against Smad4 did not affect their ability to induce osteoclastogenesis. In contrast, MDA-MB-231 factors induced phosphorylation of p38 and ERK1/2, and pharmacological inhibitors against p38 (SB203580) and MEK1/2 (PD98059) impeded the osteoclastogenic effects of cancer-derived factors. Neutralizing antibodies against TGFβ attenuated p38 activation, whereas activation of ERK1/2 was shortened in duration, but not decreased in amplitude. ERK1/2 phosphorylation induced by cancer-derived factors was blocked by MEK1/2 inhibitor, but not by Ras (manumycin A) or Raf (GW5074) inhibitors. Inhibition of protein kinase Cα using Gö6976 prevented both ERK1/2 phosphorylation and osteoclast formation in response to MDA-MB-231-derived factors. Using microspectrofluorimetry of fura-2-AM-loaded osteoclast precursors, we have found that cancer-derived factors, similar to RANKL, induced sustained oscillations in cytosolic free calcium. The calcium chelator BAPTA prevented calcium elevations and osteoclast formation in response to MDA-MB-231-derived factors. Thus, we have shown that breast cancer-derived factors induce osteoclastogenesis through the activation of calcium/protein kinase Cα and TGFβ-dependent ERK1/2 and p38 signaling pathways.

Rapamycin inhibits osteolysis and improves survival in a model of experimental bone metastases

Breast cancer metastasis to bone results in pain, pathological fractures and hypercalcemia. Activation of osteoclasts is critical for the formation of osteolytic lesions by metastasizing tumors. Although the potent drugs, zoledronic acid and Denosumab were introduced, the presence of resistant or intolerant cases necessitated the continued search of osteoclast-targeting treatments. Rapamycin acts through the mTOR pathway, which is important for osteoclast formation. Mouse mammary carcinoma 4T1 cells were injected into the tibia of balb/c mice. Rapamycin treatment significantly decreased the osteoclast population and osteolysis associated with experimental metastases. Our data indicate the benefit of rapamycin in treating metastases-associated osteolytic disease.

Breast cancer cells inhibit spontaneous and bisphosphonate-induced osteoclast apoptosis

Breast cancer metastasizes to bone where it stimulates formation of bone-resorbing osteoclasts. Bisphosphonates constitute an important treatment for osteolytic metastases. The goal of this study was to assess the effects of soluble factors produced by breast cancer cells on osteoclast survival and responsiveness to bisphosphonates. Osteoclasts derived from the murine monocytic cell line RAW264.7 or from primary mouse bone marrow were cultured for 24-48 h untreated, with 10% conditioned media (CM) from human (MDA-MB-231) or mouse (4T1) metastatic breast carcinoma cells, or with a pro-survival factor RANKL. Cancer-derived factors maintained osteoclast survival at the levels comparable to those observed with RANKL. Alendronate (10⁻⁴M) or pamidronate (10⁻⁷M) induced osteoclast apoptosis in untreated and, to a smaller extent, in RANKL-treated cultures, resulting in a significant decrease in osteoclast number and size, induction of caspase-3 cleavage and up-regulation of BIM. In the presence of cancer-derived factors, bisphosphonates were ineffective in inducing osteoclast apoptosis, resulting in only modest decrease in osteoclast numbers and not in size. MDA-MB-231 CM prevented bisphosphonate-induced cleavage of caspase-3 and up-regulation of BIM. MCSF-neutralizing antibody attenuated the effect of MDA-MB-231 CM by ~50%, but could not fully restore osteoclast responsiveness to alendronate. Inhibition of phospholipase C (PLC)-γ interfered with MDA-MB-231-induced down-regulation of BIM and prevented anti-apoptotic action of cancer-derived factors on osteoclasts. Our data suggest that factors produced by the metastatic breast cancer cells promote osteoclast survival and block the apoptotic effect of bisphosphonates in MCSF and PLC-dependent manner, potentially compromising bisphosphonate effectiveness in the bone metastasis setting.