Biomedical subjects

Jussi Koivunen

Publications and source records attributed to Jussi Koivunen.

12 recordsLinked to original sources

PubMed2006

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts.

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Protein kinase C (PKC) family in cancer progression.

PKC family consist of a number of serine-threonine kinases which are divided into three groups based on their activating factors. PKCs have been linked to carcinogenesis since PKC activators can act as tumor promoters. Furthermore, functional studies have suggested that PKCs play a role in the carcinogenesis and maintenance of malignant phenotype. Potentiation of malignant phenotype may be mediated by activation of selective PKC isoenzymes or through altered isoenzyme expression profile compared to the originating tissue. Activation of PKCalpha and beta isoenzymes have often been linked to malignant phenotype while PKCdelta is thought to mediate anti-cancer effects. This review will focus on the regulation and significance of PKC isoenzymes to cancer progression.

Animals↗

PubMed2006

Heterogeneity of cellular proliferation within transitional cell carcinoma: correlation of protein kinase C alpha/betaI expression and activity.

A total of 18 histological samples containing both transitional cell carcinoma (TCC) and normal urothelial epithelium were analyzed for protein kinase C (PKC)-alpha and -betaI expression, and for their phosphorylated substrates. The results showed an increased expression of PKC-alpha in 13 out of 18 samples and -betaI in 11 out of 18 TCC samples when compared with normal urothelium. In addition, 11 out of 18 of the TCC tumors displayed heterogeneous expression of the PKC isoenzymes, with different levels of immunosignal in different areas of the tumor. Within the same sample, areas of highest PKC isoenzyme expression also showed highest classical PKC activity, as estimated by immunodetection of phosphorylated forms of PKC substrates. The areas of highest expression of PKC-alpha and/or -betaI isoenzymes showed also the highest number of cells positive for Ki67, an indicator of proliferation. Immunofluorescence and Western blotting demonstrated that in cultured TCC cells, PKC-alpha was located in the cytoplasm, whereas PKC-betaI was located primarily in the nucleus as a 65-kDa fragment and in the cytoplasm as a full-size 79-kDa protein. Our results indicate that increased expression of PKC-alpha and -betaI leads to increased total classical PKC kinase activity and suggest that increased activity of the isoenzymes plays a role in accelerated growth of TCC. Furthermore, these results suggest that even in carcinoma tissue, PKC expression and activity are under strict control.

Carcinoma, Transitional Cell↗

PubMed2005

NF1 tumor suppressor in epidermal wound healing with special focus on wound healing in patients with type 1 neurofibromatosis.

Type 1 neurofibromatosis syndrome (NF1) has been linked with mutations of the NF1 gene which encodes tumor suppressor neurofibromin, a regulator of Ras-MAPK signaling. In human epidermis, keratinocytes express NF1 tumor suppressor and it may have a distinctive function in these cells during wound healing, such as regulating Ras activity. NF1 expression was first studied during the epidermal wound healing using suction blister method. NF1 gene expression increased both in hypertrophic and migrating zones of the healing epidermis, and also in dermal fibroblasts underneath the injury. This prompted us to study epidermal wound healing in NF1 patients. Wound healing efficiency was evaluated 4 days after blister induction by clinical, physiological and histological methods. Epidermal wound healing was equally effective in NF1 patients and healthy controls. In addition, dermal wound healing appears to function normally in NF1 patients based on retrospective and follow-up study of biopsy scars. Furthermore, the healing wounds were analyzed immunohistochemically for cell proliferation rate and Ras-MAPK activity. Neither epidermal keratinocytes nor dermal fibroblasts showed difference in the cell proliferation rate or Ras-MAPK activity between NF1 patients and controls. Interestingly, NF1 patients displayed increased cell proliferation rate and Ras-MAPK activity in periarteriolar tissue underneath the wound. The results of the study suggest that epidermal wound healing is not markedly altered in NF1 patients. Furthermore, NF1 protein seems not to have an important function as a Ras-MAPK regulator in epidermal keratinocytes or dermal fibroblasts but instead appears to be regulator of Ras-MAPK signaling in vascular tissues.

Cell Proliferation↗

PubMed2005

The effect of extracellular calcium concentration on calcium-mediated cell signaling in NF1 tumor suppressor-deficient keratinocytes.

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes from healthy volunteers and patients with type 1 neurofibromatosis (NF1) in calcium-depleted and in low calcium culture medium. In previous studies, we found evidence that mutations of the NF1 tumor suppressor gene can lead to altered calcium-mediated cell signaling in keratinocytes cultured in the presence of a high extracellular calcium concentration. The present study demonstrated that the differences between normal and NF1 keratinocytes were dependent on extracellular calcium concentration. Specifically, when keratinocytes were exposed to thapsigargin under calcium-depleted culture conditions the subsequent increase in free intracellular calcium concentration was moderate in NF1 keratinocytes compared to controls. The finding indicates lowered endoplasmic calcium stores in NF1 which may also in part explain the reduced activation signal for capacitative calcium influx and the wound-induced intracellular Ca2+ transient observed in NF1 keratinocytes maintained in culture medium containing 0.05 mM calcium. The differences between control and NF1 keratinocytes were most pronounced when the cells were cultured in the presence of a high (1.8 mM) calcium concentration. Since elevated extracellular calcium levels induce keratinocytes to form cellular contacts and lead to terminal differentiation, markedly aberrant responses of NF1 keratinocytes in the presence of a high calcium concentration may help to explain previous findings on impaired formation of cellular junctions and differentiation in NF1 deficient cells.

Adult↗

PubMed2004

Protein kinase C alpha/beta inhibitor Go6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells.

Changes in activation balance of different protein kinase C (PKC) isoenzymes have been linked to cancer development. The current study investigated the effect of different PKC inhibitors on cellular contacts in cultured high-grade urinary bladder carcinoma cells (5637 and T24). Exposure of the cells to isoenzyme-specific PKC inhibitors yielded variable results: Go6976, an inhibitor of PKCalpha and PKCbeta isoenzymes, induced rapid clustering of cultured carcinoma cells and formation of an increased number of desmosomes and adherens junctions. Safingol, a PKCalpha inhibitor, had similar but less pronounced effects. In contrast, a PKCdelta inhibitor, rottlerin, had an opposite effect on cell clustering and caused dissociation of cell junctions. A broad-spectrum PKC inhibitor bisindolylmaleimide I did not have any apparent effect on the morphology of the cultures or on the number of cell junctions. Additional studies with Go6976 demonstrated that inhibition of PKCalpha and beta isoenzymes induced translocation of beta1-integrin from the cell-matrix junctions and that beta4-integrin was translocated to face the culture substratum. Go6976 was also highly effective in inhibiting migration of carcinoma cells and inhibited invasion through artificial basement membrane. Our results on urinary bladder carcinoma cells emphasize that Go6976 is a potential anticancer drug due to its effects on cell-cell and cell-matrix junctions, migration, and invasion. Furthermore, the results may be explained by changes in PKC activation balance promoted by inhibition of PKCalpha/beta.

Actins↗

PubMed2004

NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos.

UNLABELLED: NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease. INTRODUCTION: Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development. MATERIALS AND METHODS: The expression of the NF1 gene was studied in normal and NF1+/- mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile. RESULTS: NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/- embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/- embryos. CONCLUSIONS: The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.

Animals↗

PubMed2003

Osteoblast recruitment from stem cells does not decrease by age at late adulthood.

This study was aimed to characterize the ability of human bone marrow mesenchymal stem cells (MSC) to differentiate into osteoblasts in vitro. Twenty-three women and 20 men at late adulthood (52-92 years of age) were selected for the study. MSCs were isolated and cultured in vitro and alkaline phosphatase (ALP) activity, secretion of amino-terminal propeptide of type I procollagen (PINP), type III procollagen (PIIINP) and osteocalcin were analyzed. Matrix mineralization was analyzed by the von Kossa staining and by calcium quantification. We found that the ALP and PINP levels compared with control increased to 2.8- and 2.9-fold, respectively, when cells were cultured for three weeks. ALP activity, PINP and calcium deposition in response to dexamethasone treatment increased by age in women and was unchanged in men. Overall our data suggests that the osteogenic potential of MSCs does not decrease by age in either women or men at late adulthood.

Age Factors↗

PubMed2003

An intact actin-containing cytoskeleton is required for capacitative calcium entry, but not for ATP-induced calcium-mediated cell signaling in cultured human keratinocytes.

BACKGROUND: The present study was focused on structural relationship between intracellular calcium stores and plasma membrane store-operated calcium channels in cultured normal and NF1 keratinocytes. MATERIAL/METHODS: Calcium mobilization induced by thapsigargin or extracellular ATP was studied in control and cytochalasin D-treated human keratinocytes. RESULTS: Treatment of keratinocytes with cytochalasin D disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. In normal control keratinocytes, thapsigargin induced a marked increase in intracellular calcium concentration ([Ca2+]i). The capacitative calcium influx of cytochalasin D-treated normal keratinocytes was significantly weaker compared to normal control cells. In normal keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i. Thus disruption of the cytoskeleton blocked thapsigargin-induced calcium mobilization, but had no effect on ATP-induced [Ca2+]i mobilization in keratinocytes. The results suggest that microfilaments play crucial role for functional capacitative Ca2+ entry in cultured keratinocytes. The cytoskeleton and calcium mediated cell signaling have been demonstrated to be abnormal in keratinocytes cultured from patients with neurofibromatosis type 1 (NF1). In NF1 keratinocytes, thapsigargin induced a slow and moderate increase in [Ca2+]i. The effect of cytochalasin D on NF1 keratinocytes was less pronounced compared to normal keratinocytes. In NF1 keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i. CONCLUSIONS: The actin microfilaments play a crucial role for functional capacitative Ca2+ entry in cultured keratinocytes, and that aberrant organization of cytoskeleton may partly explain altered calcium-mediated cell signaling in NF1.

Actins↗

PubMed2002

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis.

BACKGROUND: NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. METHODS: The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. RESULTS: The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8-14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with alpha-tubulin or actin of the cytoskeleton, or with adherens junction proteins. CONCLUSIONS: These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes.

Adult↗

PubMed2002

Altered calcium-mediated cell signaling in keratinocytes cultured from patients with neurofibromatosis type 1.

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

Adenosine Triphosphate↗

PubMed2002

NF1 tumor suppressor mRNA is targeted to the cell-cell contact zone in Ca(2+)-induced keratinocyte differentiation.

SUMMARY: We have previously shown that NF1 (type 1 neurofibromatosis) p21ras GTPase-activating tumor suppressor protein undergoes major relocalization during the formation of cell-cell junctions in differentiating keratinocytes in vitro. This prompted us to study the distribution of NF1 mRNA under the same conditions by in situ hybridization. In differentiating keratinocytes, the NF1 mRNA signal intensified within the cell cytoplasm within the first 0.5 to 2 hours after induction of cellular differentiation. First, the hybridization signal was evenly distributed throughout the cytoplasm. Subsequently, NF1 mRNA was gradually polarized to the cellular periphery at the side of cell-cell junctions and finally disappeared. Reappearance of NF1 mRNA was found in migrating keratinocytes forming a bilayered culture. Disruption of microfibrillar cytoskeleton, but not microtubules, caused a marked change in the subcellular distribution of NF1 mRNA. This data may suggest that intact actin microfilaments are essential for transport of NF1 mRNA to the cell periphery. This is the first study demonstrating that NF1, or any tumor suppressor mRNA, belongs to a rare group of mRNAs not targeted to free polysomes or ribosomes of the rough endoplasmic reticulum. This finding recognizes a potential way for post-transcriptional modification of NF1 expression.

Adult↗