H. Kalervo Väänänen

Biochemical Markers of Bone Metabolism and Prediction of Fracture in Elderly Women

We studied the ability of various markers of bone turnover to predict fracture in 1040 randomly recruited 75‐year‐old women. A total of 178 of the women sustained at least one fracture during follow‐up (mean, 4.6 years). In elderly women, TRACP5b and urinary fragments of osteocalcin are promising new markers for prediction of fracture, in particular, vertebral fracture.

Osteoclast lineage and function

Osteoclasts are members of the monocyte/macrophage lineage and are formed by cellular fusions from their mononuclear precursors. Their differentiation is regulated by a number of other cells and their products, especially by RANKL and M-CSF. The resorbing osteoclasts are polarized and show specific plasma membrane domains. Polarization and bone resorption need a continuous membrane trafficking and modulation of the cytoskeleton. The most characteristic feature of osteoclasts is their unique capacity to dissolve crystalline hydroxyapatite by targeted secretion of HCl into the extracellular resorption lacuna. Organic matrix is degraded by enzymes like cathepsin K and the degradation products are transcytosed through the cell for secretion. Dissolution of hydroxyapatite releases large amounts of soluble calcium, phosphate and bicarbonate. Removal of these ions apparently involves the vesicular pathways and direct ion transport via different ion exchangers, channels and pumps. Detailed molecular knowledge of osteoclast differentiation and function has helped us to identify several target molecules and develop specific treatments to inhibit pathological bone resorption in various skeletal diseases.

Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis

Carbonic anhydrase III (CA III; EC 4.2.1.1) is a cytoplasmic enzyme that exhibits a relatively low carbon dioxide hydratase activity. It is expressed at a very high level in skeletal muscle, where physical exercise has been shown to increase free radical production. In this work we show the effect of overexpression of CA III on cellular response to oxidative stress. Rat CA III cDNA was transfected to NIH/3T3 cells, which have no endogenous CA III expression. The isolated clones expressed CA III mRNA and protein. The protein was localized to cytoplasm and nuclei. Compared to parental cells, transfected cells showed lower basal oxidized state as judged by measurement of intracellular reactive oxygen species (ROS) using fluorescent dye and an image analysis system. Addition of exogenous H2O2 to cells induced a rapid increase of ROS in control but not in CA III overexpressing cells. Association of this phenomenon with CA III expression was further confirmed by showing that overexpression of CA II could not prevent H2O2‐stimulated increase of ROS. In proliferation assays, CA III overexpressing cells grew faster and were more resistant to cytotoxic concentrations of H2O2 than control cells. After a 16 h exposure to oxidative stress, the number of apoptotic cells was also reduced in transfectants. Our results suggest that CA III functions as an oxyradical scavenger and thus protects cells from oxidative damage. A lower level of free radicals in CA III overexpressing cells may also affect growth signaling pathways.—Räisänen, S. R., Lehenkari, P., Tasanen, M., Rahkila, P., Härkönen, P. L., Väänänen, H. K. Carbonic anhydrase III protects cells from hydrogen peroxide‐induced apoptosis. FASEB J. 13, 513–522 (1999)

Osteocytes inhibit osteoclastic bone resorption through transforming growth factor-β: Enhancement by estrogen*

Osteocytes are the most abundant cells in bone and distributed throughout the bone matrix. They are connected to the each other and to the cells on the bone surface. Thus, they may also secrete some regulatory factors controlling bone remodeling. Using a newly established osteocyte‐like cell line MLO‐Y4, we have studied the interactions between osteocytes and osteoclasts. We collected the conditioned medium (CM) from MLO‐Y4 cells, and added it into the rat osteoclast cultures. The conditioned medium had no effect on osteoclast number in 24‐h cultures, but it dramatically inhibited resorption. With 5, 10, and 20% CM, there was 25, 39, and 42% inhibition of resorption, respectively. Interestingly, the inhibitory effect was even more pronounced, when MLO‐Y4 cells were pretreated with 10−8 M 17‐β‐estradiol. With 5, 10, and 20% CM, there was 46, 51, and 58% of inhibition. When the conditioned medium was treated with neutralizing antibody against transforming growth factor‐β (TGF‐β), the inhibitory effect was abolished. This suggests that osteocytes secrete significant amounts of TGF‐β, which inhibits bone resorption and is modulated by estrogen. RT–PCR and Western blot analysis show that in MLO‐Y4 cells, the prevalent TGF‐β isoform is TGF‐β3. We conclude that osteocytes have an active, inhibitory role in the regulation of bone resorption. Our results further suggest a novel role for TGF‐β in the regulation of communication between different bone cells and suggest that at least part of the antiresorptive effect of estrogen in bone could be mediated via osteocytes. J. Cell. Biochem. 85: 185–197, 2002.

Mesenchymal stem cells.

About 40 years ago Friedenstein described stromal cells in the bone marrow that were spindle shaped and proliferate to form colonies. These cells attach to plastic and are able to differentiate under defined in vitro conditions into multiple cell types present in many different tissues, e.g. osteoblasts, chondroblasts, adipocytes, etc. Later on these cells, obtained from postnatal bone marrow, were called mesenchymal stem cells (MSC) or stromal stem cells. Recently the presence of somewhat similar cells has been demonstrated in many other tissues too. In spite of extensive attempts to characterize these cells we are still lacking definitive in vivo markers of MSC although retrospective functional data strongly support the existence of common adult stem cells that have the capacity to differentiate along various specific differentiation lineages. Since MSC can be rather easily isolated from the bone marrow and can also be expanded in vitro they have become a prime target for researchers of tissue regeneration. These cells have now been extensively used for transplantation experiments in animals and also for some therapeutic trials in humans. However, much new research is needed to learn enough on the molecular mechanisms of MSC differentiation to evaluate their full capacity for tissue regeneration.About 40 years ago Friedenstein described stromal cells in the bone marrow that were spindle shaped and proliferate to form colonies. These cells attach to plastic and are able to differentiate under defined in vitro conditions into multiple cell types present in many different tissues, e.g. osteoblasts, chondroblasts, adipocytes, etc. Later on these cells, obtained from postnatal bone marrow, were called mesenchymal stem cells (MSC) or stromal stem cells. Recently the presence of somewhat similar cells has been demonstrated in many other tissues too. In spite of extensive attempts to characterize these cells we are still lacking definitive in vivo markers of MSC although retrospective functional data strongly support the existence of common adult stem cells that have the capacity to differentiate along various specific differentiation lineages. Since MSC can be rather easily isolated from the bone marrow and can also be expanded in vitro they have become a prime target for researchers of tissue regeneration. These cells have now been extensively used for transplantation experiments in animals and also for some therapeutic trials in humans. However, much new research is needed to learn enough on the molecular mechanisms of MSC differentiation to evaluate their full capacity for tissue regeneration.

Estrogen and Testosterone Use Different Cellular Pathways to Inhibit Osteoclastogenesis and Bone Resorption

Using human peripheral blood CD14+ osteoclast precursors, we show that testosterone directly inhibits osteoclast formation and bone resorption at physiological concentrations. Instead, estrogen has no direct effects, whereas its action seems to be mediated through osteoblasts by producing osteoprotegerin. Both estrogen and testosterone acts through their cognate receptors.

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.

HRT and exercise: effects on bone density, muscle strength and lipid metabolism. A placebo controlled 2-year prospective trial on two estrogen-progestin regimens in healthy postmenopausal women

OBJECTIVES To evaluate the effect of 1- or 3-monthly sequential combinations of estradiol valerate (E2V) and medroxyprogesterone acetate (MPA) on menopausal symptoms, bone density, muscle strength and lipid metabolism in postmenopausal women. METHODS Changes in bone mineral density (BMD), isometric muscle strength, serum lipids and climacteric symptoms were evaluated in 78 women, 49-55 years of age, with a spontaneous menopause 0.5-3 years earlier. Treatment group I received 2 mg E2V tablets for 11 days, followed by 2 mg E2V + 10 mg MPA for 10 days and placebo for an additional 7 days; treatment group II received 2 mg E2V for 70 days, 2 mg E2V + 20 mg MPA for 14 days, and placebo for 7 days. The placebo group received placebo continuously for 24 months. Each group was further randomised to exercise and non-exercise subgroups. RESULTS Both hormone regimens significantly reduced menopausal symptoms, and prevented equally well the decrease of BMD both in the lumbar spine and proximal femur. A positive effect of exercise on BMD was observed in the placebo group. No synergistic effect of exercise and estrogen on BMD could be shown. Both hormone regimens increased the isometric strength of back extensor muscles. Serum total and LDL cholesterol decreased during the first year with both estrogen regimens. CONCLUSIONS Estrogen-progestin regimens were equally effective in the control of menopausal symptoms and preventing bone loss, increasing muscle strength and lowering serum cholesterol.

Serum TRACP 5b is a useful marker for monitoring alendronate treatment: comparison with other markers of bone turnover

We studied clinical performance of serum TRACP 5b and other bone turnover markers, including S‐CTX, U‐DPD, S‐PINP, S‐BALP, and S‐OC, for monitoring alendronate treatment. TRACP 5b had higher clinical sensitivity, area under the ROC curve, and signal‐to‐noise ratio than the other markers.

Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-term...