Helena Kaija

Intracellular Machinery for Matrix Degradation in Bone-Resorbing Osteoclasts†

In osteoclasts, TRACP co‐localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K.

O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

Enzymes as modulators in malignant transformation

Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17beta-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ERalpha had independent prognostic significance. Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.

Tartrate-resistant bone acid phosphatase: large-scale production and purification of the recombinant enzyme, characterization, and crystallization.

Tartrate‐resistant acid phosphatase (TRAP) is an enzyme expressed in bone‐resorbing osteoclasts and certain tissue macrophages in human tissues. The functions of TRAP in biological systems are not known. Elucidation of the three‐dimensional structure of the active site could yield important information about the physiological substrate(s) of the enzyme. We have produced recombinant rat bone TRAP using a baculovirus expression vector system. The production was scaled up to a 30‐l bioreactor, and a method of purification in large scale was developed. The enzyme is composed of one 34 kDa polypeptide chain. Trypsin digestion resulted in a preparation where two subunits of ∼23 kDa and ∼16 kDa appeared after disulfide reduction. Trypsin digestion activated the enzyme. We generated monoclonal antibodies against recombinant TRAP. One of the selected antibodies detected the 23 kDa subunit in Western blotting. The reduced and oxidized forms of the enzyme could be separated by Mono‐S cation‐exchange chromatography. Crystals of TRAP have been obtained with ammonium sulfate/polyethylene glycol as precipitant. They belong to space group P212121 or P21212 with unit cell dimensions a = 57.2 Å, b = 69.5 Å, and c = 87.2 Å and diffract to at least 2.2 Å resolution. A packing density value of 2.55 Å3/Da is consistent with one subunit in the asymmetric unit.

Theoretical investigations of prostatic acid phosphatase

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB‐2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide–PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide AceDNLpYYWDNH2 from ErbB‐2(1197–1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C‐terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide AceNLYpYWDQNH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb‐2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO  43− state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop–loop contact in the PAP dimer to be of importance in cooperativity. Proteins 2005.

Tartrate-resistant acid phosphatase 5B circulates in human serum in complex with α2-macroglobulin and calcium

Tartrate-resistant acid phosphatase (TRACP) is an enzyme with unknown biological function. In addition to its acid phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS) at neutral pH. Two forms of TRACP circulate in human serum, macrophage-derived TRACP 5a and osteoclast-derived TRACP 5b. Here we have studied the circulating forms of the osteoclast-derived TRACP 5b in rat and human serum. In human serum, TRACP 5b circulates in a large complex that contained alpha2M and calcium. On the contrary, rat serum TRACP 5b circulates as a free molecule. Formation of the TRACP 5b complex in vitro decreased significantly the ROS generating activity of TRACP 5b without affecting its phosphatase activity. These results suggest that the complex formation may be necessary to eliminate the formation of the harmful ROS in the neutral pH of serum.

Increased type I collagen synthesis in victims of sudden cardiac death due to idiopathic myocardial fibrosis

Abstract Aims. Idiopathic myocardial fibrosis (IMF) was observed to be the most prevalent autopsy finding in the victims of sudden cardiac death (SCD) under the age of 40 years in the FinGesture cohort. To elucidate further the mechanisms of IMF, we examined the collagen composition from the myocardial samples taken from the victims of IMF-associated SCD. Methods. Eighteen cases with IMF as a cause of death, confirmed by autopsy, were selected for the analysis. Controls (n = 27) included were cases in whom no cardiac or non-cardiac disease could be found as a cause of unexpected death at autopsy. In addition to conventional histological examination, immunohistochemical staining of procollagens I and III (PINP and PIINP), mature collagen III (IIINTP), and the cross-linked collagen I degradation product (ICTP) were performed. Results. Increased accumulation of PINP was observed in the fibrotic tissue of the IMF cases in comparison with control samples. In contrast, type III collagen was not as frequently expressed in the fibrotic areas. Conclusion. Myocardial accumulation of PINP in the victims of IMF-associated SCD indicates increased type I collagen synthesis. Future studies on the role of circulating type I collagen biomarkers are needed to study further the implications of the described association.

Stress-specific responses of p21 expression: Implication of transcript variant p21 alt-a in long-term hypoxia

p21 (CDKN1A, Cip1, Waf1) is a cyclin‐dependent kinase inhibitor capable of causing cell cycle arrest or promoting cell cycle transit as well as acting as a regulator of apoptosis. In this study, we analyzed the effects of various antemortem conditions on p21 protein level and expression profiles of known p21 transcript variants in human heart tissue. The selected death cause groups were: non‐cardiac, hypothermia, acute ischemia, and chronic hypoxia. Immunohistochemical staining of p21 in cardiac myocytes could be observed only in hypothermia death cases, in which the mRNA expression of the most abundant variant, p21V1, also exceeded that in other death cause groups. Cytoplasmic localization of p21 protein in vascular smooth muscle cells together with substantially increased expression of cardioprotective Pim‐1 especially in chronic hypoxia, but in acute ischemia and hypothermia as well, indicate change of p21 function from cell cycle arrest to promotion of proliferation and cell survival in these cases. In chronic hypoxia deaths the expression of variant p21 alt‐a was highly pronounced whereas the expression of variant p21B was low. In chronic hypoxia deaths the expression of p53 was substantially higher compared to the other groups, being a potential regulator of p21 alt‐a expression. In acute ischemia deaths increased expression of variant p21B, suggested to be proapoptotic in several cell lines, was observed. Our results suggest a role for variant p21 alt‐a in hypoxia and for variant p21B in acute myocardial ischemia. The known cardioprotective aspect of hypothermia might come from an increased p21 protein level. J. Cell. Biochem. 113: 544–552, 2012.

Hypothermia and Rewarming Induce Gene Expression and Multiplication of Cells in Healthy Rat Prostate Tissue

Prostate cancer has been extensively studied, but cellular stress responses in healthy prostate tissue are rarely investigated. Hypothermia is known to cause alterations in mRNA and protein expressions and stability. The aim of this study was to use normal rat prostate as a model in order to find out consequences of cold exposure and rewarming on the expressions of genes which are either members or functionally/structurally related to erythroblastic leukemia viral oncogene B (ErbB) signaling pathway. Relative mRNA expressions of amphiregulin (AMR), cyclin D1 (CyD1), cyclin-dependent kinase inhibitor 1A (p21), transmembrane form of the prostatic acid phosphatase (PAcP), thrombomodulin (TM) and heat shock transcription factor 1 (HSF1) in rat ventral prostate were quantified in mild (2 or 4.5 h at room temperature) and severe (2 or 4.5 h at +10°C) hypothermia and in rewarming after cold exposure (2 h at +10°C followed by 2 h at room temperature or 3 h at +28°C). AMR protein level, apoptotic Bcl-2 associated X protein to B-cell CLL/lymphoma 2 (Bax/Bcl-2) mRNA ratio and proliferative index Ki-67 were determined. 4.5-h mild hypothermia, 2-h severe hypothermia and rewarming increased expression of all these genes. Elevated proliferation index Ki-67 could be seen in 2-h severe hypothermia, and the proliferation index had its highest value in longer rewarming with totally recovered normal body temperature. Pro-apoptotic tendency could be seen in 2-h mild hypothermia while anti-apoptosis was predominant in 4.5-h mild hypothermia and in shorter rewarming with only partly recovered body temperature. Hypothermia and following rewarming promote the proliferation of cells in healthy rat prostate tissue possibly via ErbB signaling pathway.

CHAPTER 10 – Acid Phosphatases

Phosphatases can be classified according to several frameworks. They can be divided into groups based on their substrate type. Nonspecific phosphatases catalyze the hydrolysis of almost any phosphate ester, whereas protein phosphatases prefer phosphoproteins or phosphopeptides as substrates. Nonspecific phosphatases can be divided into alkaline phosphatases and acid phosphatases based on their optimal pH for catalysis. One obvious difference among phosphatases is the presence or absence of metal ion cofactors. Acid phosphatases have wide substrate specificity and some of them catalyze transphosphorylations between phosphoesters and alcohols. Nonspecific acid phosphatases recycle phosphate in metabolic reactions. Protein phosphatases are a structurally miscellaneous group of enzymes that remove phosphate groups that have been attached to amino acid residues of proteins by protein kinases. Protein phosphatases have different mechanisms of action, subcellular localization, and substrate specificity with varied demands for optimal pH. Protein phosphatases comprise two groups based upon their substrate specificity. Protein–tyrosine phosphatases prefer to remove phosphate from tyrosine residue, and serine/threonine protein phosphatases from serine or threonine residue. The PTPase group includes dual specificity phosphatases, which are capable of hydrolyzing phosphorylated tyrosine, serine, and threonine, and phosphorylated lipids. Protein phosphatases regulate signal transduction pathways. Approximately 30% of intracellular proteins are subject to reversible protein phosphorylation.