Altered Notch Signaling in Developing Molar Teeth of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Deficient Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with neuroprotective and neurotrophic effects. This suggests its influence on the development of teeth, which are, similarly to the nervous system, ectoderm and neural crest derivatives. Our earlier studies have shown morphological differences between wild-type (WT) and PACAP-deficient mice, with upregulated sonic hedgehog (SHH) signaling in the lack of PACAP. Notch signaling is a key element of proper tooth development by regulating apoptosis and cell proliferation. In this study, our main goal was to evaluate the possible effects of PACAP on Notch signaling pathway. Immunohistochemical staining was performed of Notch receptors (Notch1, 2, 3, 4), their ligands [delta-like protein (DLL)1, 3, 4, Jagged1, 2], and intracellular target molecules [CSL (CBF1 humans/Su (H) Drosophila/LAG1 Caenorhabditis elegans transcription factor); TACE (TNF-α converting enzyme), NUMB] in molar teeth of 5-day-old WT, and homozygous and heterozygous PACAP-deficient mice. We measured immunopositivity in the enamel-producing ameloblasts and dentin-producing odontoblasts. Notch2 receptor and DLL1 expression were elevated in ameloblasts of PACAP-deficient mice compared to those in WT ones. The expression of CSL showed similar results both in the ameloblasts and odontoblasts. Jagged1 ligand expression was elevated in the odontoblasts of homozygous PACAP-deficient mice compared to WT mice. Other Notch pathway elements did not show significant differences between the genotype groups. The lack of PACAP leads to upregulation of Notch pathway elements in the odontoblast and ameloblast cells. The underlying molecular mechanisms are yet to be elucidated; however, we propose SHH-dependent and independent processes. We hypothesize that this compensatory upregulation of Notch signaling by the lack of PACAP could represent a salvage pathway in PACAP-deficient animals.

Disturbed spermatogenic signaling in pituitary adenylate cyclase activating polypeptide-deficient mice.

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Structural and morphometric comparison of the molar teeth in pre-eruptive developmental stage of PACAP-deficient and wild-type mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Okadaic acid-induced inhibition of protein phosphatase 2A enhances chondrogenesis in chicken limb bud micromass cell cultures.

The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H-thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that protein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal organisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

Primary structure of human matrilin-2, chromosome location of the MATN2 gene and conservation of an AT-AC intron in matrilin genes.

We isolated full-length cDNA clones for human matrilin-2, an oligomeric protein, which forms filamentous networks in the extracellular matrices of various tissues. The human matrilin-2 precursor is encoded by a 4.0-kb mRNA, it consists of 956 amino acids and shows 93% similarity to the mouse protein. Out of the two von Willebrand factor type A-like domains, the 10 epidermal growth factor-type modules, one unique sequence and the oligomerization module, the first A domain is the most conserved. RT-PCR demonstrated wide expression of the gene in human cell lines of fibroblastic or epithelial origin. Alternative splicing affected only 19 amino acids in a 75-moiety-long segment, unique to matrilin-2. Isolation and analysis of the 3' end of the gene revealed that the reason for alternative splicing is alternative 3' splice site selection. Further, we identified in the human matrilin-2 gene a U12 type AT-AC intron between the last two exons encoding the oligomerization domain. We mapped the matrilin-2 gene (MATN2) by fluorescence in situ hybridization at chromosome position 8q22.

Consecutive appearance of coagulation factor XIII subunit A in macrophages, megakaryocytes, and liver cells during early human development.

Thirty embryonic and fetal samples were investigated to study the appearance and characteristics of factor XIII subunit A (FXIIIA)-containing cells in the course of human development. Samples were either vacuum-embedded in paraffin for staining FXIIIA by a sensitive biotin-streptavidin system or snap-frozen for double-labeling studies to characterize FXIIIA-containing cells. FXIIIA appeared as early as the fifth gestational week in yolk sac samples in stellate-shaped cells. Nonparenchymal cells in liver samples showed intense labeling for FXIIIA from the sixth week of gestation. The relative amount of FXIIIA-containing cells rapidly diminished up to the 13th gestational week. When characterized, the majority of these cells proved to be KiM7-positive macrophages, while GPIb (CD42b)-labeled cells accounted for less than 10% of FXIIIA-positive cells. Liver cells did not show any staining for FXIIIA in first trimester samples. The earliest liver specimen showing FXIIIA was at the 20th week, when FXIIIA appeared in some liver cells, particularly in those surrounding the central veins. In bone marrow smears, FXIIIA-positive cells started to appear at week 10 in the clavicles and increased in number in subsequent stages of development. Intracellular FXIIIA was distributed between GPIb-, RFD7-, and KiM7-positive cells. The results indicate that, apart from liver cells, at least three different cell populations (KiM7+ RFD7+ GPIb-, KiM7- RFD7- GPIb-, and KiM7- RFD7- GPIb+) contain FXIIIA in the early phase of human development. We conclude that FXIIIA appears very early during human development and is detectable in both extra- and intraembryonic hematopoietic organs. Intracellular FXIIIA in early human development is distributed between different macrophages and megakaryocytes, and by week 20, it appears in liver cells as well.