IgG4-positive plasma cells in Hashimoto thyroiditis: IgG4-related disease or inflammation-related IgG4-positivity?

Despite the interest of researchers in IgG4-related disease (IgG4-RD), many questions still remain unanswered regarding the thyroid gland. We aimed to clarify the relationship between IgG4-positive plasma cells and the histopathological pattern in the Hashimoto thyroiditis (HT) in a Finnish series. HT specimens (n = 280) were retrieved from the Department of Pathology, Fimlab Laboratories. After re-evaluation, 82 (29%) cases (72 females and 10 males, 52 ± 17 years) with significant fibrosis were selected. CD38, IgG and IgG4 positivity in plasma cells was evaluated by immunohistochemistry. Adjusted IgG4-positive plasma cells per HPF > 20 and IgG4- to IgG-positive plasma cell ratio > 30% were adopted as threshold criteria and related to other morphological features. IgG4-positive HT group included 13 cases (15% from fibrotic HT, 4.6% from all HT, 50 ± 15 years, 11 females) with adjusted HPF count 30 ± 5 (23-40) IgG4-positive cells. IgG4-positivity significantly correlated with the presence of lobulation, oncocytic metaplasia and certain type of fibrosis, fibrosis spread outside the gland, lymphocytes/plasma cells epithelial penetration, the predominance of microfollicles and follicular atrophy in the present study. Despite the persisting uncertainty whether HT is IgG4-RD, HT with IgG4-positive plasma cells is histopathologically distinct entity with some geographic variability.

Expression of neuroendocrine differentiation markers in lethal metastatic castration-resistant prostate cancer.

Neuroendocrine differentiation (NED) is a common phenomenon in prostate cancer, and it has been associated with poor prognosis in some studies of primary prostate cancer. Incidence and patterns of NED in metastatic prostate cancer sites have not been examined widely. In this study, we studied expression of three commonly used markers of NED (chromogranin A, neuron specific enolase and synaptophysin) in 89 metastases from 31 men that died of castration-resistant prostate cancer and underwent rapid autopsy, and in 89 hormone-naïve primary tumors removed by radical prostatectomy. In addition, we examined NED association with androgen receptor, ERG and Ki-67 expression in metastatic tumor sites. Morphologically, 1 of 31 cases was classified as small cell carcinoma, and the remaining 30 were classified as usual prostate adenocarcinoma using a recently proposed classification of prostate cancers with NED. Metastases showed more expression of neuron specific enolase and synaptophysin compared to prostatectomies (6.3% of cells vs. 1.0%, p < 0.001 and 4.0% vs. 0.4%, p < 0.001, respectively). At least focal expression of one of the markers was seen in 78% of metastases. Strong expression was relatively uncommon, seen in 3/89 (chromogranin A), 8/89 (neuron specific enolase), and 5/89 (synaptophysin) metastases. Expression of chromogranin A and synaptophysin correlated with each other (r = 0.64, p < 0.001), but expression of neuron specific enolase did not correlate with the two other markers. Extent of NED varied significantly between different metastatic sites in individual patients. Absent androgen receptor expression was associated with strong expression of chromogranin A (p = .02) and neuron specific enolase (p = .02), but not with focal expression of any marker. No clear association was found between expression of NE markers and ERG or Ki-67. In conclusion, NED is a common and heterogeneous phenomenon in metastatic, castration-resistant prostate cancer. NED is more often present in castration-resistant prostate cancer compared to hormone-naïve disease, and it is associated with androgen receptor negativity. More research is needed to understand significance of NED in the progression of prostate cancer.

Feasibility of Prostate PAXgene Fixation for Molecular Research and Diagnostic Surgical Pathology: Comparison of Matched Fresh Frozen, FFPE, and PFPE Tissues.

Advances in prostate cancer biology and diagnostics are dependent upon high-fidelity integration of clinical, histomorphologic, and molecular phenotypic findings. In this study, we compared fresh frozen, formalin-fixed paraffin-embedded (FFPE), and PAXgene-fixed paraffin-embedded (PFPE) tissue preparation methods in radical prostatectomy prostate tissue from 36 patients and performed a preliminary test of feasibility of using PFPE tissue in routine prostate surgical pathology diagnostic assessment. In addition to comparing histology, immunohistochemistry, and general measures of DNA and RNA integrity in each fixation method, we performed functional tests of DNA and RNA quality, including targeted Miseq RNA and DNA sequencing, and implemented methods to relate DNA and RNA yield and quality to quantified DNA and RNA picogram nuclear content in each tissue volume studied. Our results suggest that it is feasible to use PFPE tissue for routine robot-assisted laparoscopic prostatectomy surgical pathology diagnostics and immunohistochemistry, with the benefit of significantly improvedDNA and RNA quality and RNA picogram yield per nucleus as compared with FFPE tissue. For fresh frozen, FFPE, and PFPE tissues, respectively, the average Genomic Quality Numbers were 7.9, 3.2, and 6.2, average RNA Quality Numbers were 8.7, 2.6, and 6.3, average DNA picogram yields per nucleus were 0.41, 0.69, and 0.78, and average RNA picogram yields per nucleus were 1.40, 0.94, and 2.24. These findings suggest that where DNA and/or RNA analysis of tissue is required, and when tissue size is small, PFPE may provide important advantages over FFPE. The results also suggest several interesting nuances including potential avenues to improve RNA quality in FFPE tissues and confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided.

Hsp27 and its expression pattern in diffusely infiltrating astrocytomas.

AIMS: Heat shock protein 27 (Hsp27) is induced by cell stress conditions. In the presence of oxidative stress it functions as an antioxidant. To study the putative expression patterns and clinical significance of Hsp27, we assessed the associations between Hsp27, R132H mutation of Isocitrate dehydrogenase1 (IDH1-R132H), Hypoxia-inducible factor subunit alpha (HIF-1 alpha), Carbonic anhydrase IX (CA IX), and patient prognosis in astrocytic gliomas. METHODS: Tissue micro-array samples of 295 grade II-IV astrocytomas were stained immunohistochemically for Hsp27, IDH1-R132H, HIF-1 alpha, and CA IX. We tested their relationship with clinicopathological features and patient survival. RESULTS: There was a significant correlation between Hsp27 expression and increasing WHO grade (p<0.001). Hsp27 expression correlated significantly with IDH1 mutation when studied within the entire cohort (p<0.001) as well as separately in WHO grade II and III tumors (p=0.006 and 0.002, respectively). IDH1 mutation and HIF-1 alpha positive staining were detected simultaneously (p<0.001). In IDH1 mutated tumors, positive HIF-1 alpha staining correlated with CA IX expression (p=0.027), whereas no such correlation was found in IDH1 non-mutated tumors. IDH1 mutation was associated with a low cell proliferation index (p=0.001) and HIF-1 alpha with increasing proliferation (p = 0.003). Hsp27 expression was associated with a shorter rate of patient survival in univariate survival analysis (p=0.001). In multivariate survival analysis, patient age, IDH1 mutation and HIF-1 alpha appeared as independent prognostic factors (p<0.000, <0.000 and 0.011 respectively) CONCLUSIONS: Hsp27 expression is associated with increasing WHO grade and patient prognosis in astrocytic gliomas. The results suggest that IDH1 mutation may have an effect on the expression pathways of Hsp27 and CA IX.

DNA from dead cancer cells induces TLR9-mediated invasion and inflammation in living cancer cells.

TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.

Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.

Formalin fixation preserves tissue morphology at the expense of macromolecule integrity. Freshly frozen samples are the golden standard for DNA and RNA analyses but require laborious deep-freezing and frozen sectioning for morphological studies. Alternative tissue stabilisation methods are therefore needed. We analysed the preservation of nucleic acids, immunohistochemical staining properties and tissue morphology in paraffin-embedded clinical tissue samples fixed with Z7, RCL2, PAXgene, Allprotect and RNAlater. Formalin-fixed and deep-frozen samples were used as controls. Immunohistochemical analyses showed good preservation of antigenicity in all except Allprotect and RNAlater-fixed samples. RNA quality, based on RNA integrity number value by Bioanalyzer, was comparable with freshly frozen samples only in PAXgene-fixed samples. According to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, RNA from PAXgene samples yielded results similar to freshly frozen samples. No difference between fixatives was seen in DNA analyses (PCR and real-time PCR). In conclusion, PAXgene seems to be superior to other molecular fixatives and formaldehyde.

Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer.

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.

Routine dual-color immunostaining with a 3-antibody cocktail improves the detection of small cancers in prostate needle biopsies.

We performed dual-color immunostaining with a 3-antibody cocktail (α-methylacyl coenzyme-A racemase, CK34betaE12, and p63) on prostate biopsies from 200 patients. Current practice (hematoxylin and eosin staining followed by dual-color immunostaining on selected cases) was compared with a protocol in which routine dual-color immunostaining was provided in all cases. In the original pathology reports, adenocarcinoma was diagnosed in 87/200 (43%) patients. Small foci interpreted as putative cancers were detected with dual-color immunostaining in 14/113 patients who were originally diagnosed with a nonmalignant lesion. All of the suggested cancerous foci were independently reevaluated by 5 pathologists. A diagnosis of adenocarcinoma was assessed by consensus in 8 cases, and atypical small acinar proliferation was diagnosed in 1 case. Consensus was not reached in 5 cases. Six of the foci reclassified as cancer were of Gleason score 3 + 3 = 6, while 2 were graded as Gleason score 4 + 4 = 8. The feasibility of routine dual-color immunostaining was also tested by analyzing the time spent on microscopic assessment. Because small, atypical lesions expressing α-methylacyl coenzyme-A racemase (blue chromogen) were easy to detect using dual-color immunostaining, the microscopic analysis of dual-color immunostaining and hematoxylin-eosin staining was faster than that of hematoxylin-eosin staining alone that was later followed by dual-color immunostaining in selected cases (median 251 seconds versus 299 seconds, P < .0001). We concluded that routine dual-color immunostaining of all prostate biopsies would produce better diagnostic sensitivity with a smaller microscopy workload for the pathologist. However, minute foci interpreted as cancer with dual-color immunostaining need to be confirmed with hematoxylin-eosin staining, and minimal criteria for a definitive diagnosis of cancer are still lacking.

Multiple molecular mechanisms underlying trastuzumab and lapatinib resistance in JIMT-1 breast cancer cells.

Trastuzumab plays an important role in breast cancer therapy. However, a significant fraction of patients do not respond to therapy or they tend to develop resistance shortly after beginning therapy. Although some resistance mechanisms have been described, it is unclear whether these mechanisms can coexist. In this study, we analyzed the resistance mechanisms in the breast cancer cell line JIMT-1, a model of intrinsic trastuzumab resistance. We compared the JIMT-1 cell line with a panel of eight HER-2 positive breast cancer cell lines. All cell lines were characterized for the phosphatidylinositol 3-kinase (PIK3CA) mutation status, expression levels of the phosphatase and tensin homolog on chromosome 10 (PTEN) and neuregulin-1 (NRG1) mRNA, HER-2 gene copy number, and protein expression. The results were correlated to the sensitivity to trastuzumab and lapatinib as well as the potency of trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) evoked by trastuzumab. JIMT-1 cells showed several co-existing drug resistance mechanisms, including an activating mutation of the PIK3CA gene, low expression of PTEN, high expression of NRG1, and relatively low expression of HER-2 receptor protein (despite gene amplification). All these features were present at variable levels in other cell lines, whereas JIMT-1 was unique in displaying all these factors at the same time. Unexpectedly, ADCC reaction by normal lymphocytes was equally strong in all HER-2 positive cell lines, without any correlation to molecular markers or direct sensitivity to the drugs. Resistance to trastuzumab and lapatinib is probably caused by several co-existing molecular mechanisms. Direct sensitivity to trastuzumab and lapatinib was not correlated with ADCC.

Dioxins interfere with differentiation of osteoblasts and osteoclasts.

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.

Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion.

Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.

Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro.

BACKGROUND: Toll-like receptor 9 (TLR9) recognizes microbial DNA. In addition to immune cells, TLR9 expression has been detected in various cancer cells. We showed recently that TLR9 agonistic CpG-oligonucleotides (CpG-ODNs) induce matrix metalloproteinase-13 (MMP-13)-mediated invasion in TLR9-expressing (TLR9(+)) breast cancer cells. We investigated here TLR9 expression and function in human prostate cancer (CaP) cells. METHODS: TLR9 expression was detected with Western blotting and immunohistochemistry. Invasion was studied with Matrigel-assays. MMP-13 was assayed with ELISA. RESULTS: Human CaP cell lines and clinical samples exhibit various levels of TLR9 expression. Treatment of TLR9(+), but not TLR9(-) CaP cells with CpG-ODNs or bacterial DNA increased their invasion, which was inhibited with chloroquine. CpG-ODN-treatment also increased MMP-13 activity and neutralizing anti-MMP-13 antibody prevented CpG-ODN-induced invasion in TLR9(+) CaP cells. Estradiol up-regulated TLR9 expression in LnCaP cells. CONCLUSIONS: TLR9-mediated invasion may represent a novel mechanism through which infections promote prostate cancer.

Effect of modified pectin molecules on the growth of bone cells.

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

Differential effects of Ca(2+) on bisphosphonate-induced growth inhibition in breast cancer and mesothelioma cells.

Bisphosphonates are widely clinically used inhibitors of bone resorption. Pre-clinical studies indicate that bisphosphonates also inhibit the growth of various cancer cells in vitro, but their in vivo anti-cancer activity varies greatly, depending on the tumor type. We compared the various cellular effects of bisphosphonates in breast cancer and mesothelioma cells, with differences in growth inhibition responses to bisphosphonate-treatment in vivo. We show that the growth inhibitory effects of nitrogen-containing bisphosphonates are significantly affected by excess Ca(2+) in a cell- and bisphosphonate-specific fashion. Furthermore, excess pyrophosphate-resembling bisphosphonates prevent nitrogen-containing-bisphosphonate-induced accumulation of unprenylated Rap1A, p38 phosphorylation and growth inhibition in human MDA-MB-231 breast cancer and mouse AB-12 mesothelioma cells. For some, but not all tested, pyrophosphate-resembling bisphosphonate: nitrogen-containing bisphosphonate combinations these results may be partially explained by the ability of the excess pyrophosphate-resembling bisphosphonates to chelate Ca(2+). In mice, subcutaneous AB-12 and MDA-MB-231 tumors exhibit positive staining for Ca(2+) minerals, as revealed with Von Kossa stainings. We further show that the AB-12 tumors accumulate significantly more of the bone scanning bisphosphonate, Tc99m-medronate, as compared with MDA-MB-231 tumors. In conclusion, our results suggest that Ca(2+) regulates the growth inhibitory effects of bisphosphonates in a target cell and drug-specific fashion. These findings may be of physiological relevance since many tumor types are calcified. They further suggest that bisphosphonates can accumulate in tumors that are growing at the visceral sites and that differences in tumor accumulation of bisphosphonates may regulate their in vivo sensitivity to these drugs.

Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity.

Toll-like receptor 9 (TLR9) recognizes microbial DNA. We show here that TLR9 protein is expressed in human breast cancer cells and clinical breast cancer samples. Stimulation of TLR9-expressing breast cancer cells with the TLR9 agonistic CpG oligonucleotides (1-10 mumol/L) dramatically increased their in vitro invasion in both Matrigel assays and three-dimensional collagen cultures. Similar effects on invasion were seen in TLR9-expressing astrocytoma and glioblastoma cells and in the immortalized human breast epithelial cell line MCF-10A. This effect was not, however, dependent on the CpG content of the TLR9 ligands because the non-CpG oligonucleotides induced invasion of TLR9-expressing cells. CpG or non-CpG oligonucleotide-induced invasion in MDA-MB-231 cells was blunted by chloroquine and they did not induce invasion of TLR9(-) breast cancer cells. Treatment of MDA-MB-231 cells with CpG or non-CpG oligonucleotides induced the formation of approximately 50-kDa gelatinolytic band in zymograms. This band and the increased invasion were abolished by a matrix metalloproteinase (MMP) inhibitor GM6001 but not by a serine proteinase inhibitor aprotinin. Furthermore, CpG oligonucleotide treatment decreased tissue inhibitor of metalloproteinase-3 expression and increased levels of active MMP-13 in TLR9-expressing but not TLR9(-) breast cancer cells without affecting MMP-8. Neutralizing anti-MMP-13 antibodies inhibited the CpG oligonucleotide-induced invasion. These findings suggest that infections may promote cancer progression through a novel TLR9-mediated mechanism. They also propose a new molecular target for cancer therapy, because TLR9 has not been associated with cancer invasiveness previously.

Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro.

BACKGROUND: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. METHODS: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. RESULTS: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. CONCLUSION: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption.

Bone resorption by aryl hydrocarbon receptor-expressing osteoclasts is not disturbed by TCDD in short-term cultures.

Polychlorinated dibenzo-p-dioxins (PCDDs) are highly toxic environmental contaminants, and 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) is the most potent dioxin. Dioxins bind specifically to the cytosolic aryl hydrocarbon receptor (AHR), which is a ligand-activated transcription factor, and a majority of toxic effects of dioxins are mediated via AHR. We have recently demonstrated that TCDD disrupts bone modeling and decreases bone mechanical strength, and that partial resistance to these effects is related to an altered transactivation domain in AHR structure. In order to better understand the effects of dioxins on bone, we studied the presence and precise localization of AHR and also the number and activity of osteoclasts after TCDD treatments. Total RNA was extracted from mixed bone cell population cultures and expression of AHR mRNA was studied using RT-PCR. Bone cells expressed a considerable amount of AHR mRNA. To see which bone cells express AHR, immunostainings were performed in primary rat bone cell cultures, pure human osteoclast cultures and histological sections from AHR knockout and wild type bones. Immunostaining revealed a strong expression of AHR both in osteoclasts and osteoblasts with an especially prominent stain in bone resorbing osteoclasts. Effects of dioxin on primary bone cells were evaluated after TCDD treatment in the pit formation assay. The activity of osteoclasts was not affected measured as the percentage of active osteoclasts and the actual area of resorbed bone. These data indicate that even though TCDD-treated bones show decreased mechanical strength and size, this is not a direct result from increased osteoclastic bone resorption.

Gap-junctional regulation of osteoclast function.

Previous research has shown that bone-resorbing osteoclasts contain connexin molecules that organize to hemichannel-forming connexons, which in turn form functional gap junctions in neighboring cells. So far only little is known about the role of gap junctions in osteoclasts. However, blocking of the gap-junctional communication inhibits bone resorption in vitro. Knockout mice deficient of Connexin-43, the major connexin in bone cells, show surprisingly little skeletal manifestations. Gap-junctional communication in osteoblasts and osteocytes is well documented and seems to be essential for the integrity of bone cells, as well as for the transfer of mechanical signals of bone loading. The role of gap junction in osteoclasts is unclear, so far, but some putative roles have been suggested, including their participation in osteoclast precursor fusion to multinucleated mature osteoclasts, communication in the bone multicellular unit in bone remodeling, osteoclast survival, and apoptosis.