Michael Hüfner

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts†

Core binding factor alpha 1 (Cbfa1) is an osteoblast‐specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic‐differentiating agents is still unclear. In this study, we assessed the effects of 1,25‐(OH)2‐D3 (D3), ascorbic acid, bone morphogenetic protein‐2 (BMP‐2), dexamethasone (Dex), and transforming growth factor‐β (TGF‐β) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT‐PCR) in an in vitro model of osteoblast differentiation. TGF‐β increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6‐fold in a time‐dependent fashion with maximum effect on day 28 (Pu2009<u20090.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time‐dependent fashion by up to 4.6‐fold (Pu2009<u20090.001). In contrast, Dex inhibited OCN gene expression levels by 68% (Pu2009<u20090.01). Treatment with BMP‐2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2‐fold increase with a maximum on day 21 (Pu2009<u20090.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5‐fold after 48 h; Pu2009<u20090.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14‐fold (Pu2009<u20090.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation. J. Cell. Biochem. 86: 348–356, 2002.

DEVELOPMENT OF THE OSTEOBLAST PHENOTYPE IN PRIMARY HUMAN OSTEOBLASTS IN CULTURE : COMPARISON WITH RAT CALVARIAL CELLS IN OSTEOBLAST DIFFERENTIATION

In rat osteoblast‐like cells, a time‐dependent sequence of growth and differentiation‐dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix‐associated proteins osteonectin and procollagen I and of the bone cell phenotype‐related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25‐(OH)2D3 stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so‐called “proliferation” as well as a similar, but lengthened, sequence for the “matrix maturation stage.” During “matrix maturation,” we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the “matrix maturation stage,” as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species‐specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system. J. Cell Biochem. 75:22–35, 1999.

Risk Profiles and Penetrance Estimations in Multiple Endocrine Neoplasia Type 2A Caused by Germline RET Mutations Located in Exon 10

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical‐risk profiles. Presentation, age‐dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4–86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon‐associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609→620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected. Hum Mutat 31:1–8, 2010.

Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases†

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)‐1, IL‐6, and TNF‐α protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin‐6 (26‐fold) and TNF‐α (84‐fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72–0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF‐α. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF‐α. In conclusion, in an in vitro‐ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF‐α seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.

Anti‐carcinoembryonic antigen antibodies versus somatostatin analogs in the detection of metastatic medullary thyroid carcinoma

Surgery is currently the only potentially curative approach in the treatment of medullary thyroid carcinoma (MTC). In many instances however, postsurgically elevated or rising plasma calcitonin and/or carcinoembryonic antigen (CEA) levels indicate persistent metastatic disease, although conventional diagnostic procedures (computed tomography (CT), magnetic resonance imaging (MRI), and invasive venous catheterization) fail to localize the responsible lesions. Recently, anti‐CEA antibodies and somatostatin analogs have shown promising results in the staging of MTC. The aim of this study was to compare the sensitivity of both methodologies, especially for the detection of occult MTC, and to assess whether there may be correlations between the scintigraphic behavior and the patients prognosis.

Reverse transcriptase/polymerase chain reaction analysis of parathyroid hormone-related protein for the detection of tumor cell dissemination in the peripheral blood and bone marrow of patients with breast cancer

Tumor cell dissemination in the bone marrow is an independent prognostic marker for relapse and survival for patients with primary breast cancer. Parathyroid-hormone-related protein (PTHrP) is expressed in most primary tumors and bone metastases of patients with breast cancer. PTHrP acts as an autocrine growth factor for breast cancer cells in vitro and there is evidence that it is especially important for osseous metastasis. For a sensitive detection of PTHrP-positive disseminated tumor cells a reverse transcriptase/polymerase chain reaction (RT/PCR) assay for PTHrP transcripts in the peripheral blood (PB) and in the bone marrow (BM) has been established. In mixing studies, the sensitivity of the reverse transcriptase/polymerase chain reaction (RT/PCR) for PTHrP was one tumor cell in 1×106 mononuclear cells. At this level of sensitivity, transcripts of PTHrP were detected in none of 30 PB samples and in 3 of 25 BM samples of healthy volunteers: there were also no transcripts of PTHrP in the PB and BM of 6 patients with benign breast lesions. The PB samples of 31 patients and the BM samples of 34 patients with predominantly early-stage breast cancer were tested for PTHrP expression along with immunocytology against cytokeratin 18 (CK18) as a standard immunological detection technique. PTHrP expression was shown in 9 of 31 patients in the PB and in 9 of 34 patients in the BM. In 30 patients, PB and BM samples were available simultaneously. There were cases of combined positive findings in the PB and the BM (4/30) and of isolated positivity in the PB (5/30) or in the BM (4/30). Compared to immunocytology, RT/PCR assay of PTHrP assay was significantly more sensitive in the peripheral blood (8/30 by RT/PCR compared to 1/30 by immunocytology). In the bone marrow there were cases of positivity for both markers (2/34), cases of isolated positivity by immunocytology for CK18 (3/34) and cases of isolated positivity for PTHrP transcripts (7/34). In conclusion the RT/PCR assay for PTHrP transcripts is a feasible and very sensitive technique for the detection of tumor cell dissemination in the PB, even in patients with early-stage breast cancer. The specificity of detection of PTHrP transcripts in the bone marrow is limited, possibly because of autochthonous expression of PTHrP in osteoblastic cells. The clinical follow-up of the subgroups of patients at risk, as defined by this assay, will show its prognostic significance for patients with breast cancer.

Paraneoplastic hypokalemia in acute myeloid leukemia: a case of renin activity in AML blast cells

Abstractu2002Hypokalemia due to renal potassium loss has frequently been observed in patients with acute myeloid leukemia (AML). The pathogenic mechanism for this hyperkaluresis is unclear. In this report we describe a patient with AML FAB M4, in whom the clinical course, the electrolyte disturbances, the serum aldosterone levels, and the diffuse hyperplasia of the adrenal cortex documented a typical case of marked secondary hyperaldosteronism. On analysis of the leukemic cells of this patient compared with normal bone marrow cells, a significant increase of renin-like activity in the cytosol of the blast cells was noted. Activation of the renin-angiotensin-aldosterone system by paraneoplastic production of renin-like activity in AML blast cells might contribute to the hypokalemia often observed in patients with acute myeloid leukemia.

Psychoendocrine sequelae of chronic testosterone deficiency

The precise role of testosterone in regulating mood, especially in alleviating depression, remains unclear although converging evidence indicates that androgens may exert antidepressant action. A model that may potentially assist in the clarification of androgen-mediated effects on mood is the study of cryptorchid men who may grow up with varying degrees of testosterone deficiency depending on the time in their life when cryptorchism is corrected. In this report, we describe a rare case of bilateral cryptorchism that did not come to the attention of the physician to implement effective substitution with testosterone until much later in adult life. The patient developed severe and suicidal depression which responded solely to testosterone. In addition, the patient experienced a delayed but accelerated puberty without any adverse events. These observations, although based on a single case, provide strong evidence that testosterone may exert powerful antidepressant action in the absence of concomitant antidepressant agents.

Coexpression of osteogenic and adipogenic differentiation markers in selected subpopulations of primary human mesenchymal progenitor cells

Knowledge of the basic mechanisms controlling osteogenesis and adipogenesis might provide new insights into the prevention of osteoporosis and age‐related osteopenia. With the help of magnetic cell sorting and fluorescence activated cell sorting (FACS), osteoblastic subpopulations of mesenchymal progenitor cells were characterized. Alkaline phosphatase (AP) negative cells expressed low levels of osteoblastic and adipocytic markers. AP positive cells expressed adipocytic markers more strongly than the AP negative cell populations, thus suggesting that committed osteoblasts exhibit a greater adipogenic potential. AP negative cells differentiated to the mature osteoblastic phenotype, as demonstrated by increased AP‐activity and osteocalcin secretion under standard osteogenic culture conditions. Surprisingly, this was accompanied by increased expression of adipocytic gene markers such as peroxisome proliferator‐activated receptor‐γ2, lipoprotein lipase and fatty acid binding protein. The induction of adipogenic markers was suppressed by transforming growth factor‐β1 (TGF‐β1) and promoted by bone morphogenetic protein 2 (BMP‐2). Osteogenic culture conditions including BMP‐2 induced both the formation of mineralized nodules and cytoplasmic lipid vacuoles. Upon immunogold electron microscopic analysis, osteoblastic and adipogenic marker proteins were detectable in the same cell. Our results suggest that osteogenic and adipogenic differentiation in human mesenchymal progenitor cells might not be exclusively reciprocal, but rather, a parallel event until late during osteoblast development. J. Cell. Biochem. 104: 1342–1355, 2008.

HLA DQ2 and/or DQ8 Is Associated With Celiac Disease–Specific Autoantibodies To Tissue Transglutaminase in Families With Thyroid Autoimmunity

TO THE EDITOR: Celiac disease (CD) is characterized by a T cell–mediated destruction of intestinal architecture that can lead to global malabsorption but presents with atypical symptoms in the majority of patients. In CD the ingestion of dietary gluten and related cereals drives intestinal inflammation and a mucosal autoimmune response to the ubiquitous enzyme tissue transglutaminase (tTG) (1, 2). Interestingly, tTG can potentiate gluten antigen presentation and immunogenicity in genetically predisposed individuals bearing the CD-predisposing major histocompatibility complex human leukocyte antigen (HLA) DQ2 or DQ8, which is found in 95% and 5% of patients, respectively (2, 3). Long-lasting CD is associated with a variety of autoimmune diseases, mainly type 1 diabetes and dermatitis herpetiformis (4). Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are the most common autoimmune disorders. They affect more than 2% of the female population in the US. These autoimmune endocrinopathies occur frequently in patients and relatives with other autoimmune diseases, such as type 1 diabetes mellitus or CD, sharing immunogenetic susceptibility loci on chromosome 6p. Thus the haplotype allele DQA1*0501 of the HLA DQ2 heterodimer DQA1*0501/ DQB1*0201, and the HLA DQ8 heterodimer DQA1*0301/ DQB1*0302, characteristic for CD patients, are also frequently found in patients with type 1 diabetes mellitus or GD (5). An association of CD with thyroid autoimmunity has been reported in patients from Italy, Finland, and the US (6–9). Because IgA autoantibodies to tTG (tTG-Ab) are a sensitive, highly specific tool for detecting atypical or subclinical CD, we measured the prevalence of these autoantibodies in patients and families with thyroid autoimmune diseases and correlated the findings with HLA DQ status. We enrolled 105 patients with thyroid autoimmune disorders (81 with GD, 24 with HT) and 95 of their first degree relatives (43 fathers, 43 mothers, and nine siblings). The diagnosis of GD was based on hyperthyroidism with thyroid stimulating hormone receptor autoantibodies, and that of HT on hypothyroidism with thyroid peroxidase autoantibodies. tTG-Ab were measured as described (10), and HLA DQA1 and DQB1 alleles determined by polymerase chain reaction (5). There was one patient with HT and known CD who also suffered from type 1 diabetes. This patient displayed high titer tTG-Ab (93 U). Another patient with HT showed a slightly elevated titer (12 U). With these two patients a prevalence of 8% among the studied patients with HT was found, whereas we did not detect tTG-Ab among patients with GD. However, five of 43 fathers (12%) and one mother (2%) of the patients with GD had slightly or clearly elevated tTG-Ab (n 5 5, 10–17 U; n 5 1, 41 U, respectively). Three of the six (50%) tTG-Ab positive parents had the HLA DQ2 specificity, as compared with only 20 of 80 (25%) of the tTG-Ab negative parents (reflecting the frequency in the normal population) (Table 1). Furthermore, these alleles were inherited by their children who had GD. In addition, HLA DQ2 and/or DQ8 were found in all five tTG-Ab positive fathers, as compared with only 17 of 38 (45%) tTG-Ab negative fathers (p 5 0.02). Remarkably, the single patient who displayed both HLA DQ2 and HLA DQ8 suffered from triple autoimmunity (type I diabetes, GD, and CD). The finding that unaffected parents of patients with autoimmune disorders transmit the susceptibility genes to their