Publications by authors named "Anke Baranowsky"

29 Publications

  • Page 1 of 1

Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFβ/CTGF Axis.

Cells 2021 Feb 18;10(2). Epub 2021 Feb 18.

Center for Experimental Medicine, Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFβ/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFβ-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells10020430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922044PMC
February 2021

Transgenic inhibition of interleukin-6 trans-signaling does not prevent skeletal pathologies in mucolipidosis type II mice.

Sci Rep 2021 Feb 11;11(1):3556. Epub 2021 Feb 11.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany.

Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-82802-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878873PMC
February 2021

Relevance of Notch Signaling for Bone Metabolism and Regeneration.

Int J Mol Sci 2021 Jan 29;22(3). Epub 2021 Jan 29.

Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany.

Notch1-4 receptors and their signaling pathways are expressed in almost all organ systems and play a pivotal role in cell fate decision by coordinating cell proliferation, differentiation and apoptosis. Differential expression and activation of Notch signaling pathways has been observed in a variety of organs and tissues under physiological and pathological conditions. Bone tissue represents a dynamic system, which is constantly remodeled throughout life. In bone, Notch receptors have been shown to control remodeling and regeneration. Numerous functions have been assigned to Notch receptors and ligands, including osteoblast differentiation and matrix mineralization, osteoclast recruitment and cell fusion and osteoblast/osteoclast progenitor cell proliferation. The expression and function of Notch1-4 in the skeleton are distinct and closely depend on the temporal expression at different differentiation stages. This review addresses the current knowledge on Notch signaling in adult bone with emphasis on metabolism, bone regeneration and degenerative skeletal disorders, as well as congenital disorders associated with mutant Notch genes. Moreover, the crosstalk between Notch signaling and other important pathways involved in bone turnover, including Wnt/β-catenin, BMP and RANKL/OPG, are outlined.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22031325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865281PMC
January 2021

Tranexamic Acid Promotes Murine Bone Marrow-Derived Osteoblast Proliferation and Inhibits Osteoclast Formation In Vitro.

Int J Mol Sci 2021 Jan 5;22(1). Epub 2021 Jan 5.

Clinic of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany.

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22010449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795046PMC
January 2021

Proinflammatory and bone protective role of calcitonin gene-related peptide alpha in collagen antibody-induced arthritis.

Rheumatology (Oxford) 2021 Apr;60(4):1996-2009

Berlin Institute of Health (BIH), Berlin, Germany.

Objectives: Calcitonin gene-related peptide alpha (αCGRP) represents an immunomodulatory neuropeptide implicated in pain perception. αCGRP also functions as a critical regulator of bone formation and is overexpressed in patients with rheumatoid arthritis (RA). In the present study, we investigated the role of αCGRP in experimental RA regarding joint inflammation and bone remodelling.

Methods: Collagen II-antibody-induced arthritis (CAIA) was induced in wild type (WT) and αCGRP-deficient (αCGRP-/-) mice. Animals were monitored over 10 and 48 days with daily assessments of the semiquantitative arthritis score and grip strength test. Joint inflammation, cartilage degradation and bone erosions were assessed by histology, gene expression analysis and µCT.

Results: CAIA was accompanied by an overexpression of αCGRP in WT joints. αCGRP-/- mice displayed reduced arthritic inflammation and cartilage degradation. Congruently, the expression of TNF-α, IL-1β, CD80 and MMP13 was induced in WT, but not αCGRP-/- animals. WT mice displayed an increased bone turnover during the acute inflammatory phase, which was not the case in αCGRP-/- mice. Interestingly, WT mice displayed a full recovery from the inflammatory bone disease, whereas αCGRP-/- mice exhibited substantial bone loss over time.

Conclusion: This study demonstrates a proinflammatory and bone protective role of αCGRP in CAIA. Our data indicate that αCGRP not only enhances joint inflammation, but also controls bone remodelling as part of arthritis resolution. As novel αCGRP inhibitors are currently introduced clinically for the treatment of migraine, their potential impact on RA progression warrants further clinical investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/rheumatology/keaa711DOI Listing
April 2021

Procalcitonin Exerts a Mediator Role in Septic Shock Through the Calcitonin Gene-Related Peptide Receptor.

Crit Care Med 2021 Jan;49(1):e41-e52

Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Objectives: Clinically, procalcitonin represents the most widely used biomarker of sepsis worldwide with unclear pathophysiologic significance to date. Pharmacologically, procalcitonin was shown to signal through both calcitonin receptor and calcitonin gene-related peptide receptor in vitro, yet the identity of its biologically relevant receptor remains unknown.

Design: Prospective randomized animal investigations and in vitro human blood studies.

Setting: Research laboratory of a university hospital.

Subjects: C57BL/6J mice and patients with post-traumatic sepsis.

Interventions: Procalcitonin-deficient mice were used to decipher a potential mediator role in experimental septic shock and identify the relevant receptor for procalcitonin. Cecal ligation and puncture and endotoxemia models were employed to investigate septic shock. Disease progression was evaluated through survival analysis, histology, proteome profiling, gene expression, and flow cytometry. Mechanistic studies were performed with cultured macrophages, dendritic cells, and gamma delta T cells. Main findings were confirmed in serum samples of patients with post-traumatic sepsis.

Measurements And Main Results: Procalcitonin-deficient mice are protected from septic shock and show decreased pulmonary inflammation. Mechanistically, procalcitonin potentiates proinflammatory cytokine expression in innate immune cells, required for interleukin-17A expression in gamma delta T cells. In patients with post-traumatic sepsis, procalcitonin positively correlates with systemic interleukin-17A levels. In mice with endotoxemia, immunoneutralization of interleukin-17A inhibits the deleterious effect of procalcitonin on disease outcome. Although calcitonin receptor expression is irrelevant for disease progression, the nonpeptide calcitonin gene-related peptide receptor antagonist olcegepant, a prototype of currently introduced antimigraine drugs, inhibits procalcitonin signaling and increases survival time in septic shock.

Conclusions: Our experimental data suggest that procalcitonin exerts a moderate but harmful effect on disease progression in experimental septic shock. In addition, the study points towards the calcitonin gene-related peptide receptor as relevant for procalcitonin signaling and suggests a potential therapeutic application for calcitonin gene-related peptide receptor inhibitors in sepsis, which warrants further clinical investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/CCM.0000000000004731DOI Listing
January 2021

Piezo1 Inactivation in Chondrocytes Impairs Trabecular Bone Formation.

J Bone Miner Res 2021 Feb 12;36(2):369-384. Epub 2020 Nov 12.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The skeleton is a dynamic tissue continuously adapting to mechanical stimuli. Although matrix-embedded osteocytes are considered as the key mechanoresponsive bone cells, all other skeletal cell types are principally exposed to macroenvironmental and microenvironmental mechanical influences that could potentially affect their activities. It was recently reported that Piezo1, one of the two mechanically activated ion channels of the Piezo family, functions as a mechanosensor in osteoblasts and osteocytes. Here we show that Piezo1 additionally plays a critical role in the process of endochondral bone formation. More specifically, by targeted deletion of Piezo1 or Piezo2 in either osteoblast (Runx2Cre) or osteoclast lineage cells (Lyz2Cre), we observed severe osteoporosis with numerous spontaneous fractures specifically in Piezo1 mice. This phenotype developed at an early postnatal stage and primarily affected the formation of the secondary spongiosa. The presumptive Piezo1 osteoblasts in this region displayed an unusual flattened appearance and were positive for type X collagen. Moreover, transcriptome analyses of primary osteoblasts identified an unexpected induction of chondrocyte-related genes in Piezo1 cultures. Because Runx2 is not only expressed in osteoblast progenitor cells, but also in prehypertrophic chondrocytes, these data suggested that Piezo1 functions in growth plate chondrocytes to ensure trabecular bone formation in the process of endochondral ossification. To confirm this hypothesis, we generated mice with Piezo1 deletion in chondrocytes (Col2a1Cre). These mice essentially recapitulated the phenotype of Piezo1 animals, because they displayed early-onset osteoporosis with multiple fractures, as well as impaired formation of the secondary spongiosa with abnormal osteoblast morphology. Our data identify a previously unrecognized key function of Piezo1 in endochondral ossification, which, together with its role in bone remodeling, suggests that Piezo1 represents an attractive target for the treatment of skeletal disorders. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.4198DOI Listing
February 2021

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Dis Model Mech 2020 11 18;13(11). Epub 2020 Nov 18.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using knockout ( ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from and knock-in ( ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.046425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687858PMC
November 2020

Effects of CGRP receptor antagonism on glucose and bone metabolism in mice with diet-induced obesity.

Bone 2021 Feb 14;143:115646. Epub 2020 Sep 14.

Clinic of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Berlin Institute of Health (BIH), Berlin, Germany. Electronic address:

The neuropeptide calcitonin gene-related peptide (CGRP) and its receptor, calcitonin receptor-like receptor (CLR) complexing with receptor activity-modifiying protein 1 (RAMP1), have been shown to be crucially involved in the pathogenesis of migraine. However, CGRP also plays a pivotal role in regulating bone turnover and was suggested to contribute to the development of the metabolic syndrome. Therefore, our study was designed to characterize the effects of CGRP antagonism on bone and glucose metabolism in a murine model of diet-induced obesity (DIO). A subcutaneous pellet releasing the CGRP receptor antagonist BIBN 4096 (BIBN; olcegepant) was implanted in WT mice with DIO. Metabolic effects were assessed through body- and organ-weights, oral glucose tolerance (oGT), serum lipids, and gene-expression studies. Bone turnover was assessed through histomorphometry of non-decalcified bone sections and analyses of bone turnover markers in serum samples. BIBN treatment did not alter body weight gain or the levels of serum lipids including triacylglycerol and cholesterol during DIO. BIBN led to a moderate improvement of oGT which was accompanied by an increased expression of stearoyl-CoA desaturase in the liver. In skeletal tissue, BIBN treatment resulted in reduced bone volume. This was explained by decreased parameters of bone formation whereas bone resorption was not affected. Our results indicate that inhibition of CGRP signaling only moderately affects glucose metabolism during DIO but significantly impairs bone formation. As novel agents blocking CGRP or its receptor are currently introduced clinically for the treatment of migraine disorders, their potential negative impact on bone metabolism requires further clinical studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2020.115646DOI Listing
February 2021

The neuropeptide calcitonin gene-related peptide alpha is essential for bone healing.

EBioMedicine 2020 Sep 24;59:102970. Epub 2020 Aug 24.

Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany. Electronic address:

Background: Impaired fracture healing represents an ongoing clinical challenge, as treatment options remain limited. Calcitonin gene-related peptide (CGRP), a neuropeptide targeted by emerging anti-migraine drugs, is also expressed in sensory nerve fibres innervating bone tissue.

Method: Bone healing following a femoral osteotomy stabilized with an external fixator was analysed over 21 days in αCGRP-deficient and WT mice. Bone regeneration was evaluated by serum analysis, µCT analysis, histomorphometry and genome-wide expression analysis. Bone-marrow-derived osteoblasts and osteoclasts, as well as the CGRP antagonist olcegepant were employed for mechanistic studies.

Findings: WT mice with a femoral fracture display increased CGRP serum levels. αCGRP mRNA expression after skeletal injury is exclusively induced in callus tissue, but not in other organs. On protein level, CGRP and its receptor, calcitonin receptor-like receptor (CRLR) complexing with RAMP1, are differentially expressed in the callus during bone regeneration. On the other hand, αCGRP-deficient mice display profoundly impaired bone regeneration characterised by a striking reduction in the number of bone-forming osteoblasts and a high rate of incomplete callus bridging and non-union. As assessed by genome-wide expression analysis, CGRP induces the expression of specific genes linked to ossification, bone remodeling and adipogenesis. This suggests that CGRP receptor-dependent PPARγ signaling plays a central role in fracture healing.

Interpretation: This study demonstrates an essential role of αCGRP in orchestrating callus formation and identifies CGRP receptor agonism as a potential approach to stimulate bone regeneration. Moreover, as novel agents blocking CGRP or its receptor CRLR are currently introduced clinically for the treatment of migraine disorders, their potential negative impact on bone regeneration warrants clinical investigation.

Funding: This work was funded by grants from the Else-Kröner-Fresenius-Stiftung (EKFS), the Deutsche Forschungsgemeinschaft (DFG), and the Berlin Institute of Health (BIH).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ebiom.2020.102970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452713PMC
September 2020

Role of c-Fos in orthodontic tooth movement: an in vivo study using transgenic mice.

Clin Oral Investig 2021 Feb 15;25(2):593-601. Epub 2020 Aug 15.

Department of Orthodontics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.

Objectives: The transcription factor c-Fos controls the differentiation of osteoclasts and is expressed in periodontal ligament cells after mechanical stimulation in vitro. However, it is unclear how c-Fos regulates orthodontic tooth movement (OTM) in vivo. The aim of this study was therefore to analyse OTM in transgenic mice with overexpression of c-Fos.

Materials And Methods: We employed c-Fos transgenic mice (c-Fos tg) and wild-type littermates (WT) in a model of OTM induced by Nitinol tension springs that were bonded between the left first maxillary molars and the upper incisors. The unstimulated contralateral side served as an internal control. Mice were analysed by contact radiography, micro-computed tomography, decalcified histology and histochemistry.

Results: Our analysis of the unstimulated side revealed that alveolar bone and root morphology were similar between c-Fos tg and control mice. However, we observed more osteoclasts in the alveolar bone of c-Fos tg mice as tartrate-resistant acid phosphatase (TRAP)-positive cells were increased by 40%. After 12 days of OTM, c-Fos tg mice exhibited 62% increased tooth movement as compared with WT mice. Despite the faster tooth movement, c-Fos tg and WT mice displayed the same amount of root resorption. Importantly, we did not observe orthodontically induced tissue necrosis (i.e. hyalinization) in c-Fos tg mice, while this was a common finding in WT mice.

Conclusion: Overexpression of c-Fos accelerates tooth movement without causing more root resorption.

Clinical Relevance: Accelerated tooth movement must not result in more root resorption as higher tissue turnover may decrease the amount of mechanically induced tissue necrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00784-020-03503-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819946PMC
February 2021

Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

J Am Soc Nephrol 2020 08 8;31(8):1796-1814. Epub 2020 Jul 8.

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms.

Methods: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis.

Results: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis.

Conclusions: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2019090960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460914PMC
August 2020

Mice Carrying a Ubiquitous R235W Mutation of Wnt1 Display a Bone-Specific Phenotype.

J Bone Miner Res 2020 09 20;35(9):1726-1737. Epub 2020 May 20.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Since a key function of Wnt1 in brain development was established early on through the generation of non-viable Wnt1-deficient mice, it was initially surprising that WNT1 mutations were found to cause either early-onset osteoporosis (EOOP) or osteogenesis imperfecta type XV (OI-XV). The deduced function of Wnt1 as an osteoanabolic factor has been confirmed in various mouse models with bone-specific inactivation or overexpression, but mice carrying disease-causing Wnt1 mutations have not yet been described. Triggered by the clinical analysis of EOOP patients carrying a heterozygous WNT1 mutation (p.R235W), we introduced this mutation into the murine Wnt1 gene to address the question of whether this would cause a skeletal phenotype. We observed that Wnt1 and Wnt1 mice were born at the expected Mendelian ratio and that they did not display postnatal lethality or obvious nonskeletal phenotypes. At 12 weeks of age, the homozygous presence of the Wnt1 mutation was associated with reduced trabecular and cortical bone mass, explained by a lower bone formation rate compared with wild-type littermates. At 52 weeks of age, we also observed a moderate bone mass reduction in heterozygous Wnt1 mice, thereby underscoring their value as a model of WNT1-dependent EOOP. Importantly, when we treated wild-type and Wnt1 mice by daily injection of parathyroid hormone (PTH), we detected the same osteoanabolic influence in both groups, together with an increased cortical thickness in the mutant mice. Our data demonstrate the pathogenicity of the WNT1-R235W mutation, confirm that controlling skeletal integrity is the primary physiological function of Wnt1, and suggest that osteoanabolic treatment with teriparatide should be applicable for individuals with WNT1-dependent EOOP. © 2020 American Society for Bone and Mineral Research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.4043DOI Listing
September 2020

Large osteocyte lacunae in iliac crest infantile bone are not associated with impaired mineral distribution or signs of osteocytic osteolysis.

Bone 2020 06 19;135:115324. Epub 2020 Mar 19.

Department of Orthopedics, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Lottestr. 59, 22529 Hamburg, Germany. Electronic address:

The enlargement of osteocyte lacunae via osteocytic osteolysis was previously detected in situations of increased calcium demand (e.g., lactation, vitamin D deficiency). However, it is unclear whether similar processes occur also in the growing infantile skeleton and how this is linked to the mineral distribution within the bone matrix. Human iliac crest biopsies of 30 subjects (0-6 months, n = 14; 2-8 years, n = 6 and 18-25 years, n = 10) were acquired. Bone microarchitecture was assessed by micro-CT, while cellular bone histomorphometry was performed on undecalcified histological sections. Quantitative backscattered electron imaging (qBEI) was conducted to determine the bone mineral density distribution (BMDD) as well as osteocyte lacunar size and density. We additionally evaluated cathepsin K positive osteocytes using immunohistochemistry. Infantile bone was characterized by various signs of ongoing bone development such as higher bone (re)modeling, lower cortical and trabecular thickness compared to young adults. Importantly, a significantly higher osteocyte lacunar density and increased lacunar area were detected. Large osteocyte lacunae were associated with a more heterogeneous bone mineral density distribution of the trabecular bone matrix due to the presence of hypermineralized cartilage remnants, whereas the mean mineralization (i.e., CaMean) was not different in infantile bone. Absence of cathepsin K expression in osteocyte lacunae indicated nonexistent osteocytic osteolysis. Taken together, we demonstrated that the overall mineralization distribution in infantile bone is not altered compared to young adults besides high trabecular mineralization heterogeneity. Our study also provides important reference values for bone microstructure, BMDD and osteocyte characteristics in infants, children and young adults. Infantile bone displays large osteocyte lacunae indicating a developmental phenomenon rather than osteocytic osteolysis. Larger osteocytes may have superior mechanosensory abilities to enable bone adaption during growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2020.115324DOI Listing
June 2020

Modeling Spontaneous Bone Metastasis Formation of Solid Human Tumor Xenografts in Mice.

Cancers (Basel) 2020 Feb 7;12(2). Epub 2020 Feb 7.

Institute of Anatomy and Experimental Morphology, University Cancer Center Hamburg, 20251 Hamburg, Germany.

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro "metastasis" assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12020385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072706PMC
February 2020

Enzyme replacement therapy in mice lacking arylsulfatase B targets bone-remodeling cells, but not chondrocytes.

Hum Mol Genet 2020 03;29(5):803-816

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104678PMC
March 2020

Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin.

J Cell Biol 2013 Feb 11;200(4):537-49. Epub 2013 Feb 11.

Department of Osteology and Biomechanics, University Medical Center Hamburg Eppendorf, Hamburg 20246, Germany.

Although Wnt signaling is considered a key regulatory pathway for bone formation, inactivation of β-catenin in osteoblasts does not affect their activity but rather causes increased osteoclastogenesis due to insufficient production of osteoprotegerin (Opg). By monitoring the expression pattern of all known genes encoding Wnt receptors in mouse tissues and bone cells we identified Frizzled 8 (Fzd8) as a candidate regulator of bone remodeling. Fzd8-deficient mice displayed osteopenia with normal bone formation and increased osteoclastogenesis, but this phenotype was not associated with impaired Wnt signaling or Opg production by osteoblasts. The deduced direct negative influence of canonical Wnt signaling on osteoclastogenesis was confirmed in vitro and through the generation of mice lacking β-catenin in the osteoclast lineage. Here, we observed increased bone resorption despite normal Opg production and a resistance to the anti-osteoclastogenic effect of Wnt3a. These results demonstrate that Fzd8 and β-catenin negatively regulate osteoclast differentiation independent of osteoblasts and that canonical Wnt signaling controls bone resorption by two different mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201207142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575535PMC
February 2013

p65-Dependent production of interleukin-1β by osteolytic prostate cancer cells causes an induction of chemokine expression in osteoblasts.

Cancer Lett 2012 Apr 20;317(1):106-13. Epub 2011 Nov 20.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany.

Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2011.11.016DOI Listing
April 2012

Control of bone formation by the serpentine receptor Frizzled-9.

J Cell Biol 2011 Mar 14;192(6):1057-72. Epub 2011 Mar 14.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.

Although Wnt signaling in osteoblasts is of critical importance for the regulation of bone remodeling, it is not yet known which specific Wnt receptors of the Frizzled family are functionally relevant in this process. In this paper, we show that Fzd9 is induced upon osteoblast differentiation and that Fzd9(-/-) mice display low bone mass caused by impaired bone formation. Our analysis of Fzd9(-/-) primary osteoblasts demonstrated defects in matrix mineralization in spite of normal expression of established differentiation markers. In contrast, we observed a reduced expression of chemokines and interferon-regulated genes in Fzd9(-/-) osteoblasts. We also identified the ubiquitin-like modifier Isg15 as one potential downstream mediator of Fzd9 in these cells. Importantly, our molecular analysis further revealed that canonical Wnt signaling is not impaired in the absence of Fzd9, thus explaining the absence of a bone resorption phenotype. Collectively, our results reveal a previously unknown function of Fzd9 in osteoblasts, a finding that may have therapeutic implications for bone loss disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201008012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063134PMC
March 2011

Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

PLoS One 2010 Apr 27;5(4):e10309. Epub 2010 Apr 27.

Institute of Osteology and Biomechanics, University Medical Center Hamburg Eppendorf, Hamburg, Germany.

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010309PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860505PMC
April 2010

Increased trabecular bone formation in mice lacking the growth factor midkine.

J Bone Miner Res 2010 Aug;25(8):1724-35

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Midkine (Mdk) and pleiotrophin (Ptn) comprise a family of heparin-binding growth factors known primarily for their effects on neuronal cells. Since transgenic mice overexpressing Ptn have been reported to display increased bone density, we have previously analyzed Ptn-deficient mice but failed to detect any abnormality of skeletal development and remodeling. Together with the finding that Mdk expression increases in the course of primary osteoblast differentiation, we reasoned that Mdk, rather than Ptn, could play a physiologic role in bone formation. Here, we show that Mdk-deficient mice display an increased trabecular bone volume at 12 and 18 months of age, accompanied by cortical porosity. Histomorphometric quantification demonstrated an increased bone-formation rate compared with wild-type littermates, whereas bone resorption was differentially affected in trabecular and cortical bone of Mdk-deficient mice. To understand the effect of Mdk on bone formation at the molecular level, we performed a genome-wide expression analysis of primary osteoblasts and identified Ank and Enpp1 as Mdk-induced genes whose decreased expression in Mdk-deficient osteoblasts may explain, at least in part, the observed skeletal phenotype. Finally, we performed ovariectomy and observed bone loss only in wild-type but not in Mdk-deficient animals. Taken together, our data demonstrate that Mdk deficiency, at least in mice, results in an increased trabecular bone formation, thereby raising the possibility that Mdk-specific antagonists might prove beneficial in osteoporosis therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jbmr.75DOI Listing
August 2010

BMP-7-induced ectopic bone formation and fracture healing is impaired by systemic NSAID application in C57BL/6-mice.

J Orthop Res 2010 Jun;28(6):785-91

Department of Trauma-, Hand-, and Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Nonsteroidal antiinflammatory drugs (NSAIDs) are known to potentially impair the fracture healing process. The aim of the present study was to determine if the impairment of bone healing by systemic NSAID application is, at least in part, due to an interaction of NSAIDs with the bone anabolic BMP-7 pathway. Therefore, we first analyzed fracture healing in control and diclofenac-treated mice, where we not only found a significant impairment of fracture healing due to diclofenac treatment as assessed by biomechanical testing and microCT imaging, but also found high coexpression of bone morphogenetic protein-7 (BMP-7) and cyclooxygenase-2 (COX-2) within the fracture callus of both groups. To experimentally address the possible interaction between BMP-7 and COX-2, we then induced ectopic bone formation in control (n = 10) and diclofenac-treated mice (n = 10) by application of BMP-7 (recombinant human OP-1, rhOP-1) into the hamstring muscles. After 20 days of treatment, each ectopic bone nodule was analyzed by contact-radiography, microCT, histology, and histomorphometry. Diclofenac application decreased the trabecular number and bone mass in the ectopic bone nodules significantly due to reduced osteoblast number and activity. These data demonstrate that the bone anabolic effect of BMP-7 and fracture healing is impaired by diclofenac application, and suggest that the potential negative impact of NSAIDs on fracture healing is, at least in part, due to interference with BMP-7 signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jor.21044DOI Listing
June 2010

Osteolytic prostate cancer cells induce the expression of specific cytokines in bone-forming osteoblasts through a Stat3/5-dependent mechanism.

Bone 2010 Feb 29;46(2):524-33. Epub 2009 Sep 29.

Center of Biomechanics and Skeletal Biology, Department of Trauma, Hand, and Reconstructive Surgery, University Medical Center Hamburg Eppendorf, Hamburg 20246, Germany.

Prostate cancer primarily metastasizes to bone, and the interaction of cancer cells with bone cells results in a local activation of bone formation and/or bone resorption. Since the cellular and molecular mechanisms underlying the development of these tumor-induced osteoblastic or osteolytic lesions are still poorly understood, we have compared the effects of two prostate cancer cell lines, osteoblastic MDA-PCa-2b cells and osteolytic PC-3 cells, on bone-forming osteoblasts. Using Affymetrix Gene Chip hybridization followed by qRT-PCR confirmation we were able to identify specific genes, including Smpd3 and Dmp1, whose expression is significantly reduced upon treatment with PC-3-conditioned medium. Moreover, we observed that PC-3-conditioned medium led to a marked induction of several cytokine genes, including Cxcl5, Cxcl12 and Tnfsf11, the latter one encoding for the osteoclast differentiation factor Rankl. Likewise, when we analyzed the effects of MDA-PCa-2b- and PC-3-conditioned medium on signal transduction in osteoblasts we did not only observe opposite effects on the canonical Wnt signalling pathway, but also a specific induction of Erk and Stat phosphorylation by PC-3-conditioned medium. Most importantly, the induction of Cxcl5, Cxcl12 and Tnfsf11 in osteoblasts by PC-3-conditioned medium was abrogated by the Stat3/5 inhibitor piceatannol, whereas the selective blockade of Stat1 and Erk activation had no effect. Together with the finding, that activated Stat3 in osteoblasts was detectable in bone biopsies from patients with osteolytic metastases, our data suggest that the Stat3/5-dependent activation of cytokine expression in osteoblasts may have a significant impact on cancer cell migration and proliferation, but also on osteoclast activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bone.2009.09.024DOI Listing
February 2010

Impaired wound healing in mice lacking the basement membrane protein nidogen 1.

Matrix Biol 2010 Jan 18;29(1):15-21. Epub 2009 Sep 18.

Department of Dermatology, University Hospital Cologne, Cologne, Germany.

Nidogens 1 and 2 are ubiquitous basement membrane (BM) components, whose interactions in particular with laminin, collagen IV and perlecan have been considered important for BM formation. Genetic deletion of either NID gene does not reveal BM alterations suggesting compensatory roles for nidogens 1 and 2. However, neurological deficits in nidogen 1 null mice, not seen in the absence of nidogen 2, also suggest isoform specific functions. To test this further, skin wound healing which requires BM reformation was studied in adult nidogen 1 deficient mice. Although re-epithelialization was not altered, the newly formed epidermis showed marked hyperproliferation and a delay in differentiation at day 10 post injury. Distinct to control wounds, there was also considerable alpha-smooth muscle actin staining in the dermis of nidogen 1 deficient wounds at this time point. Further, laminin deposition and distribution of the beta1 and beta4 integrin chains were also significantly altered whereas the deposition of other BM components, including nidogen 2, was unchanged. Surprisingly, these differences between control and mutant wounds at day 10 post wounding did not affect the ultrastructural appearance of the dermo-epidermal BM suggesting a non-structural role for nidogen 1 in wound repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2009.09.004DOI Listing
January 2010

Impaired gastric acidification negatively affects calcium homeostasis and bone mass.

Nat Med 2009 Jun;15(6):674-81

Department of Trauma, Hand, and Reconstructive Surgery, Center for Biomechanics and Skeletal Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Activation of osteoclasts and their acidification-dependent resorption of bone is thought to maintain proper serum calcium levels. Here we show that osteoclast dysfunction alone does not generally affect calcium homeostasis. Indeed, mice deficient in Src, encoding a tyrosine kinase critical for osteoclast activity, show signs of osteopetrosis, but without hypocalcemia or defects in bone mineralization. Mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells, have the expected defects in gastric acidification but also secondary hyperparathyroidism and osteoporosis and modest hypocalcemia. These results suggest that alterations in calcium homeostasis can be driven by defects in gastric acidification, especially given that calcium gluconate supplementation fully rescues the phenotype of the Cckbr-mutant mice. Finally, mice deficient in Tcirg1, encoding a subunit of the vacuolar proton pump specifically expressed in both osteoclasts and parietal cells, show hypocalcemia and osteopetrorickets. Although neither Src- nor Cckbr-deficient mice have this latter phenotype, the combined deficiency of both genes results in osteopetrorickets. Thus, we find that osteopetrosis and osteopetrorickets are distinct phenotypes, depending on the site or sites of defective acidification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.1963DOI Listing
June 2009

Basement membranes in skin are differently affected by lack of nidogen 1 and 2.

J Invest Dermatol 2008 Sep 20;128(9):2259-67. Epub 2008 Mar 20.

Department of Dermatology, University of Cologne, Cologne, Germany.

Nidogens have been proposed to play a key role in basement membrane (BM) formation. However, recent findings using genetic approaches and organotypic coculture models demonstrated distinct tissue requirements thus changing the classical view of BM assembly. Toward this end, we have analyzed the dermo-epidermal junction and the microvasculature in skin of nidogen-deficient mice for their BM composition and structural assembly. Histology of nidogen double-null embryos at embryonic day (E)18.5 revealed overall normal skin morphology with a regularly differentiated epidermis. However, in the dermis, numerous erythrocytes had extravasated out of the microvasculature. Residual composition and ultrastructure of the dermo-epidermal BM are not altered in the absence of nidogens, demonstrating that the deposition of laminin, collagen IV, and perlecan occurs and allows cutaneous BM formation. In contrast, in capillaries, BM formation is severely impaired in the absence of nidogens, showing an irregular, patchy distribution and a dramatically reduced deposition of collagen IV, perlecan, and particularly laminin-411. Ultrastructure revealed thin fragile walls in the small blood vessels next to the epidermis, completely lacking a distinct endothelial BM. In summary, our results indicate that in skin the laminin composition of the various BMs determines whether nidogens are required for their assembly and stabilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2008.65DOI Listing
September 2008

Lack of nidogen-1 and -2 prevents basement membrane assembly in skin-organotypic coculture.

J Invest Dermatol 2007 Mar 28;127(3):545-54. Epub 2006 Sep 28.

Department of Dermatology, University of Cologne, Cologne, Germany.

Nidogens are considered as classical linkers joining laminin and collagen IV networks in basement membranes (BMs); however, recent genetic approaches have suggested that nidogens function in a tissue-specific and developmental context. Thus, in mice lacking both nidogen-1 and -2 heart and lung were severely affected, causing neonatal death. Furthermore, in various locations, extravasation of erythrocytes was observed implying microvascular defects. Mice expressing solely either isoform, had a functional BM, although nidogen-2 binds with lower affinity to the laminin gamma1 chain. Having previously blocked BM formation by interfering with nidogen-1 binding to laminin in skin-organotypic cocultures, here we investigated the roles of nidogen-1 and -2 in this model. For that purpose, human HaCaT cells were grown in three-dimensional cocultures on collagen matrices containing murine fibroblasts of varying nidogen deficiency. As with our experiments blocking laminin-nidogen interaction, lack of both nidogens completely prevented BM deposition and ultrastructural assembly of BM and hemidesmosomes, although other BM proteins remained detectable at comparable levels with no signs of degradation. Supplementation by recombinant nidogen-1 or -2 restored these structures, as shown by immunofluorescence and electron microscopy, confirming that in this system nidogen-2 is equivalent to nidogen-1, and both can promote the development of a functional BM zone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.jid.5700562DOI Listing
March 2007

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice.

Mol Cell Biol 2005 Aug;25(15):6846-56

Department of Dermatology, University of Cologne, 50924 Cologne, Germany.

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.25.15.6846-6856.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190363PMC
August 2005

Impaired sphingomyelinase activity and epidermal differentiation in atopic dermatitis.

J Invest Dermatol 2004 Jun;122(6):1423-31

Department of Dermatology, University of Kiel, Kiel, Germany.

A defective permeability barrier leads to the penetration of environmental allergens into the skin and initiates immunological reactions and inflammation crucially involved in the pathogenesis of atopic dermatitis (AD). Decreased stratum corneum ceramide content may cause the defect in permeability barrier function consistently found in AD. Acid and neutral sphingomyelinase (A- and N-SMase) generate ceramides with structural and signal transduction functions in epidermal proliferation and differentiation. We determined epidermal SMase activities, DNA synthesis, involucrin, loricrin, filaggrin, and keratin expression in lesional and non-lesional skin of AD patients. We found decreased epidermal A-SMase activity in lesional and non-lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function. N-SMase activity was reduced in non-lesional skin and more significantly reduced in lesional skin, correlating with impaired expression of cornified envelope proteins and keratins, important for skin barrier function. Changes in involucrin, loricrin, filaggrin, keratin K 5 (basal) and K 16 (proliferation associated) were noticed in non-lesional and lesional skin, whereas changes in K 10 (suprabasal), K 6 (proliferation associated), and K 17 (inflammation associated) were found only in lesional skin. In summary, reduction in SMase-generating ceramides and impaired differentiation are involved in the defective barrier function found in AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.0022-202X.2004.22621.xDOI Listing
June 2004