Although the cure rate for cutaneous squamous cell carcinoma is high, the diverse spectrum of squamous cell carcinoma has made it difficult for early diagnosis, particularly the aggressive tumors that are highly associated with mortality. Therefore, molecular markers are needed as an adjunct to current staging methods for diagnosing high-risk lesions, and stratifying those patients with aggressive tumors. To identify such biomarkers, we have examined a comprehensive set of 200 histologically defined squamous cell carcinoma and normal skin samples by using a combination of microarray, QRT-PCR and immunohistochemistry analyses. A characteristic and distinguishable profile including matrix metalloproteinase (MMP) as well as other degradome components was differentially expressed in squamous cell carcinoma compared with normal skin samples. The expression levels of some of these genes including matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 10 (MMP10), parathyroid hormone-like hormone (PTHLH), cyclin-dependent kinase inhibitor 2A (CDKN2A), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), FBJ osteosarcoma oncogene (FOS), interleukin 6 (IL6) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) were significantly differentially expressed (P≤0.02) in squamous cell carcinoma compared with normal skin. Furthermore, based on receiver operating characteristic analyses, the mRNA and protein levels of MMP1 are significantly higher in aggressive tumors compared with non-aggressive tumors. Given that MMPs represent the most prominent family of proteinases associated with tumorigenesis, we believe that they may have an important role in modulating the tumor microenvironment of squamous cell carcinoma.

The gene coding for rat parathyroid hormone-like peptide (PTHLH) was previously assigned to rat chromosome 2 (Hendy et al., 1988). We reexamined this assignment. According to our results, the gene is on rat chromosome 4. Taking into account the known localizations of the KRAS2 (Kras-2) oncogene and the PTHLH gene, this assignment strongly suggests that a synteny group is conserved on rat chromosome 4, mouse chromosome 6, and human chromosome 12.

Studies were done on the analysis of biological processes in the same high expression (fold change ≥ 2) PTHLH-activated feedback negative regulation-mediated apoptosis gene ontology (GO) network of human hepatocellular carcinoma (HCC) compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues [hepatitis B virus (HBV) or hepatitis C virus (HCV) infection]. We proposed PTHLH-activated network that upstream included the regulation of apoptosis, signal transduction resulting in induction of apoptosis, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, negative regulation of centriole replication, negative regulation of fatty acid biosynthesis, negative regulation of Wnt receptor signaling pathway, anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, apoptosis, induction of apoptosis, and negative regulation of phosphorylation. Downstream-network negative regulation of peptidase activity, anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, apoptosis, induction of apoptosis and negative regulation of phosphorylation, as a result of coupling upstream negative regulation of fatty acid biosynthesis and Wnt receptor signal to downstream peptidase activity-induced apoptosis in HCC. Our hypothesis was verified by the different PTHLH-activated feedback negative regulation-mediated apoptosis GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. PTHLH coupling upstream negative regulation of fatty acid biosynthesis and Wnt receptor signal to downstream peptidase activity-induced apoptosis network was constructed that upstream BRCA1, DKK1, BUB1B activated PTHLH, and downstream PTHLH-activated CST6, BUB1B, NTN1, PHLDA2 in HCC from GEO data set using gene regulatory network inference method and our programing.

Brachydactyly includes shortening of digits due to abnormal development of phalanges, metacarpals, or both. It can occur either as an isolated malformation or with other anomalies as part of many congenital syndromes. It is included as one of the dysostosis groups affecting the limbs in the nosology and classification of genetic skeletal disorders. However, brachydactyly usually shows a high degree of phenotypic variability. In this study, we successfully identified a novel heterozygous mutation of the parathyroid hormone-like hormone (PTHLH) gene by exome sequencing in a Chinese pedigree with brachydactyly and short stature. The PTHLH gene encodes a parathyroid hormone-related protein (PTHrP) that is involved in the regulation of endochondral bone development, and mutations in this gene cause the type E form of brachydactyly. The mutation p.L15R occurs at a hydrophobic core region of the signal peptide, suggesting that this variation probably changes the signal peptide cleavage site at the in silico prediction. Further in vitro functional analysis showed that this mutation can lead to the retention of an N-terminal signal peptide fragment after the nascent proteins are translated.

Parathyroid hormone-like hormone gene (PTHLH) and its receptor, parathyroid hormone/ parathyroid hormone-like hormone receptor 1 (PTHR1), play a role in epithelial mesenchymal interactions during growth and differentiation of different tissues and anatomic structures, including teats. Therefore, PTHLH and PTHR1 were evaluated as functional candidate genes for their effects on number and shape of teats in pigs. In particular, focus was on the occurrence and number of inverted teats, the most frequent and economically relevant teat developmental defect in pigs. For this purpose, association and linkage of the PTHLH gene and the PTHR1 gene with inverted teat defect and the total number of teats and inverted teats were studied in an experimental Duroc and Berlin Miniature pig (DUMI) population. Polymorphism C1819T of PTHR1 was significantly associated with inverted teat phenotype (p = 0.014), total number of teats (p = 0.047) and was close to significance with the number of inverted teats (p = 0.078). Polymorphism C375T of PTHLH was close to significance with the inverted teat phenotype (p = 0.122) and showed no significant association with the total number of teats (p = 0.621) and the number of inverted teats (p = 0.256) in the DUMI population. Association analyses were also performed for combined effects of PTHLH and PTHR1 in order to address potential interaction, however, revealed no indication of effects of interaction. The function, position and the association shown here promote PTHR1 as a candidate gene for number of teats and in particular for affection by and number of inverted teats.

Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f) Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation.

OBJECTIVE

Role and timing of frameshift mutations during carcinogenesis in hereditary nonpolyposis colorectal cancer have not been examined. This study was designed to clarify the relationship between frameshift mutations and clinicopathologic features in colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.

METHODS

Thirty-one colorectal cancers from patients with hereditary nonpolyposis colorectal cancer at different clinicopathologic stages were analyzed for frameshift mutation in 18 genes.

RESULTS

The frameshift mutations of the ACVR2 and PTHLH genes were found to have an extremely high frequency (94-100 percent) in all pathologic stages, and mutation of the MARCKS gene also was high (94 percent) in Dukes B and C cancers. These frequencies were higher than the frequency of TGFbetaRII gene inactivation (64-88 percent). Mutations of the hMSH3, TCF4, CASP5, RIZ, RAD50, and MBD4 genes were comparatively frequent (>35 percent) in all stages. Frequencies of inactivation of the MARCKS, BAX, IGFIIR, and PTEN genes were significantly higher in Dukes B and C cancers than in Dukes A cancer (P < 0.05). The number of accumulated frameshift mutations was larger in Dukes B and C cancers (9.4) than in Dukes A cancer (6.8) (P = 0.003).

CONCLUSIONS

The present data suggest that the disruption of the transforming growth factor-beta super-family signaling pathway by the alteration of the ACVR2 and/or TGFbetaRII genes and the disruption of antiproliferative function by the PTHLH gene alteration contribute to the development of early colorectal cancer. Moreover, the further accumulation of alterations in the MARCKS, BAX, IGFIIR, and PTEN genes seem to be associated with progression from early to advanced colorectal cancer from patients with hereditary nonpolyposis colorectal cancer.

The mouse parathyroid hormone-like hormone Pthlh(Pro) and Pthlh(Thr) variants are linked with susceptibility and resistance to skin carcinogenesis of Car-S and Car-R mice, respectively, and with in vitro effects (Oncogene, 19: 5324-5328, 2000). We have identified an additional Pthlh variant, consisting of Thr and three amino-acid changes in the C-terminus (Pthlh(SerAspTyr)), carried by an evolutionarily distant Mus spretus (SPRET/Ei) inbred strain. When transfected into NCI-H520 tumor cells, this Pthlh(SerAspTyr) variant did not stimulate tumor growth in nude mice. Analysis of cell adhesion, migration, and invasion patterns of Pthlh(Pro)-, Pthlh(Thr)-, and Pthlh(SerAspTyr)-transfected NCI-H520 cells revealed a 1.5-fold decrease in adhesion efficiency on both collagen type I and Matrigel, and a 5-6-fold increase in migration capability in Pthlh(Pro) transfectants as compared to nontransfected, vector-transfected, Pthlh(Thr)-, or Pthlh(SerAspTyr)-transfected cells. These findings suggest that the cancer modifier effects of the mouse Pthlh gene are mediated by differential cell adhesion and migration effects of PTHrP variants.

The mouse parathyroid hormone-like hormone (Pthlh) gene encodes three allelic variants characterized by amino acid substitutions that are associated with susceptibility (Pthlh(Pro)) or resistance (Pthlh(Thr) and Pthlh(SerAspTyr)) to two-stage skin carcinogenesis and to modulation of cell migration in vitro in transfected human cancer cells. cDNA microarray hybridization analysis of 8473 transcript clones revealed a similar gene expression profile for the Pthlh(Thr) and Pthlh(SerAspTyr) alleles but a distinct pattern for the Pthlh(Pro) allele, suggesting an association between a specific gene expression profile and biological function of the Pthlh alleles. Some of the genes modulated by the Pthlh alleles, e.g., ANXA1, CCL2, FN1 and TFF3, play a role in cell migration and may represent candidate targets for this Pthlh function. Our study demonstrates the potential usefulness of gene expression profiling of genetic variants for the functional characterization of candidate cancer modifier genes.

Parathyroid hormone-like hormone (PTHLH, MIM 168470) is a humoral factor, structurally and functionally related to parathyroid hormone, which mediates multiple effects on chondrocyte, osteoblast and osteoclast function. Mutations and copy number imbalances of the PTHLH locus and in the gene encoding its receptor, PTHR1, result in a variety of skeletal dysplasias including brachydactyly type E, Eiken syndrome, Jansen metaphyseal chondrodysplasia and Blomstrand type chondrodysplasia. Here we describe three individuals with duplications of the PTHLH locus, including two who are mosaic for these imbalances, leading to a hitherto unrecognized syndrome characterized by acro-osteolysis, cortical irregularity of long bones and metadiaphyseal enchondromata.

Can RNA sequencing of human cumulus cells (CC) reveal molecular pathways involved in the physiology of reproductive aging?

Senescent but not young CC activate gene pathways associated with hypoxia and oxidative stress.

Shifts in socioeconomic norms are resulting in larger numbers of women postponing childbearing. The reproductive potential is sharply decreased with aging, and the reasons are poorly understood. Since CCs play an integral role in oocyte maturation and direct access to human oocytes is limited, we used whole transcriptome analysis of these somatic cells to gain insights into the molecular mechanisms playing a role in follicular senescence.

Twenty CC samples (from a total of 15 patients) were obtained from oocytes of either male factor or egg donor patients. RNA sequencing and bioinformatic tools were used to identify differentially expressed genes between CCs from seven aged and eight young patients (<35 (years old) y.o. vs >40 y.o.). Quantitative-PCR and immunoflourescent staining were used for validation.

RNA sequencing identified 11 572 genes expressed in CC of both age cohorts, 45 of which were differentially expressed. In CC collected from patients >40 y.o., genes involved in the hypoxia stress response (NOS2, RORA and NR4A3), vasculature development (NR2F2, PTHLH), glycolysis (RALGAPA2 and TBC1D4) and cAMP turnover (PDE4D) were significantly overexpressed when compared with CC of patients younger than 35 y.o.

This study focused almost exclusively on assessing the genetic differences in CC transcriptome between young and older women. These genetic findings were not fully correlated with embryonic development and clinical outcome.

Our data provide a new hypothesis-follicular hypoxia-as the main mechanism leading to ovarian follicular senescence and suggest a link between cumulus cell aging and oocyte quality decay. If specific molecular findings of hypoxia would be confirmed also in oocytes, genetic platforms could screen CC for hypoxic damage and identify healthier oocytes. Protocols of ovarian stimulation in older patients could also be adjusted to diminish oocyte exposure time to hypoxic follicles.

GEO accession number: GSE81579 STUDY FUNDING AND COMPETING INTERESTS: Funded in part by EMD Serono Grant for Fertility Innovation (GFI).

Parathyroid Hormone-Like Hormone (PTHLH) is an autocrine/paracrine ligand that is up-regulated in head and neck squamous cell carcinoma (HNSCC). However, the cellular function and regulatory mechanism in HNSCC remains obscure. We investigated the clinical significance of PTHLH in HNSCC patients, and verified the role of RUNX2/PTHLH axis, which is stimulated HNSCC cell growth. In patients, PTHLH is a poor prognosis marker. PTHLH expression lead to increasing the cell proliferation potential through an autocrine/paracrine role and elevating blood calcium level in Nod-SCID mice. In public HNSCC microarray cohorts, PTHLH is found to be co-expressed with RUNX2. Physiologically, PTHLH is regulated by RUNX2 and also acting as key calcium regulator. However, elevations of calcium concentration also increased the RUNX2 expression. PTHLH, calcium, and RUNX2 form a positive feedback loop in HNSCC. Furthermore, ectopic RUNX2 expression also increased PTHLH expression and promoted proliferation potential through PTHLH expression. Using cDNA microarray analysis, we found PTHLH also stimulated expression of cell cycle regulators, namely CCNA2, CCNE2, and CDC25A in HNSCC cells, and these genes are also up-regulated in HNSCC patients. In summary, our results reveal that PTHLH expression is a poor prognosis marker in HNSCC patients, and RUNX2-PTHLH axis contributes to HNSCC tumor growth.

OBJECTIVE

Intrahepatic cholangiocarcinoma (ICC) is an aggressive tumor with a high fatality rate. It was recently found that parathyroid hormone-like hormone (PTHLH) was frequently overexpressed in ICC compared with non-tumor tissue. This study aimed to elucidate the underlying mechanisms of PTHLH in ICC development.

METHODS

The CCK-8 assay, colony formation assays, flow cytometry and a xenograft model were used to examine the role of PTHLH in ICC cells proliferation. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. Luciferase reporter, chromatin immunoprecipitation (ChIP) and DNA pull-down assays were used to verify the transcription regulation of activating transcription factor-2 (ATF2).

RESULTS

PTHLH was significantly upregulated in ICC compared with adjacent and normal tissues. Upregulation of PTHLH indicated a poor pathological differentiation and intrahepatic metastasis. Functional study demonstrated that PTHLH silencing markedly suppressed ICC cells growth, while specific overexpression of PTHLH has the opposite effect. Mechanistically, secreted PTHLH could promote ICC cell growth by activating extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways, and subsequently upregulated ATF2 and cyclinD1 expression. Further study found that the promoter activity of PTHLH were negatively regulated by ATF2, indicating that a negative feedback loop exists.

CONCLUSIONS

Our findings demonstrated that the ICC-secreted PTHLH plays a characteristic growth-promoting role through activating the canonical ERK/JNK-ATF2-cyclinD1 signaling pathways in ICC development. We identified a negative feedback loop formed by ATF2 and PTHLH. In this study, we explored the therapeutic implication for ICC patients.

Our aim was to characterize clinical findings and familial associations, and to examine candidate genes for disease-causing mutations in a cohort of children suffering from primary osteoporosis without features of osteogenesis imperfecta. Patients with osteoporosis and their nuclear families were studied. Medical history was reviewed. Calcium homeostasis parameters were measured and spinal radiographs obtained. BMD was determined by DXA for patients, parents and siblings. LRP5, LRP6, and PTHLH genes were sequenced. Twenty-seven patients (14 males) from 24 families were recruited. Median age at presentation was 10.1 years (range 3.3-15.6 years). One-third of the children had at least one parent with a BMD below the expected range for age. LRP5, LRP6, and PTHLH showed no causative mutations. Four polymorphisms in LRP5 were overrepresented in patients; the minor allele frequency of Q89R, V667M, N740N, and A1330V was significantly higher than in controls. Age of onset, clinical severity, and inheritance patterns are variable in children with primary osteoporosis. Several patients had evidence suggestive of familial transmission. The underlying genetic factors remain to be elucidated.

BACKGROUND

Gastrectomy may disturb the body's mineral homeostasis, with osteopenia and osteoporosis being among the late outcomes. Parathyroid hormone-like hormone (PTHLH) was detected in rat gastric enterochromaffin-like (ECL) cells in 2005, and some researchers suggested that it was the hypothetical hormone gastrocalcin that is believed to lead to osteoporosis.

OBJECTIVE

Our objective was to learn whether PTHLH is expressed in human gastric ECL cells and to form a basic understanding about the relationship between PTHLH and gastrin.

METHODS

We collected normal human gastric mucosa specimens and serum samples from 28 patients.

RESULTS

RT-PCR and immunohistochemical analysis demonstrated weak expression of PTHLH in ECL cells at the RNA and protein levels. A low level of PTHLH expression was also found in the serum. Serum gastrin did have a significant positive correlation with the relative ratio of PTHLH mRNA to β-actin levels in gastric mucosa (rs=0.569, p=0.002).

CONCLUSIONS

This indicates that PTHLH has a low signal expression in human gastric ECL cells and that serum gastrin levels correlate with PTHLH RNA levels in gastric mucosa. Further work is needed to evaluate the functional role of PTHLH in ECL cells and to determine whether PTHLH is gastrocalcin.

One of the key regulators of endochondral ossification is Indian hedgehog (Ihh), which acts as a long-range morphogen in the developing skeletal elements. Previous studies have shown that the distribution and signaling activity of Ihh is regulated by the concentration of the extracellular glycosaminoglycan heparan sulfate (HS). An essential step during biosynthesis of HS is the epimerization of D-glucuronic to L-iduronic acid by the enzyme glucuronyl C5-epimerase (Hsepi or Glce). Here we have investigated chondrocyte differentiation in Glce deficient mice and found increased regions of proliferating chondrocytes accompanied by a delayed onset of hypertrophic differentiation. In addition, we observed increased expression levels of the Ihh target genes Patched1 (Ptch1) and Parathyroid hormone related peptide (Pthrp; Parathyroid hormone like hormone (Pthlh)) indicating elevated Ihh signaling. We further show that Ihh binds with reduced affinity to HS isolated from Glce(-/-) mice. Together our results strongly indicate that not only the level, but also the structure of HS is critical in regulating the distribution and signaling activity of Ihh in chondrocytes.

BACKGROUND

Human bone marrow mesenchymal stem cells (hBM-MSC) have the ability of differentiating into chondrocytes and osteoblasts. miR-182-5p promotes osteoclastogenesis and bone metastasis by up-regulating the expression of parathyroid hormone-like hormone (PTHLH). However, the function of miR-182-5p in chondrogenesis is still unknown.

METHODS

Mimic or inhibitor of miR-182-5p were used to upregulate or knock-down miR-182-5p expression respectively. We analyzed chondrogenesis by Safranin O staining and Blyscan™ Sulfated Glycosaminoglycan Assay. Immunohistochemistry, real-time PCR and western bolts were used to detect related makers.

RESULTS

miR-182-5p overexpression inhibited chondrogenesis. Dual-luciferase reporter assay indicated that PTHLH was one of the target genes of miR-182-5p. Further studies showed that miR-182-5p overexpression down-regulated the expression of SOX-9 and COL2A1, but up-regulated COL1A1 and COL10A1. Consistently, miR-182-5p knock-down had the opposite effects. This effect of miR-182-5p in BM-MSCs can be rescued by PTHLH overexpression.

CONCLUSIONS

miR-182-5p may play a negative role in chondrogenesis by down-regulating PTHLH.

OBJECTIVE

Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs.

METHODS

Embryonic day 14 rat lungs were cultured with FGF-10 (0-250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry.

RESULTS

Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh (sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively.

CONCLUSIONS

Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC and POD are known to regulate branching morphogenesis; however, potential roles of CLIC-4 and MKP-3 in lung branching morphogenesis remain to be investigated. FGF-10 may also function in regulating branching morphogenesis or inducing cystic lung growth by inhibiting Wnt2/β-catenin signaling.

BACKGROUND

Parathyroid hormone-like hormone (PTHLH) is abundantly expressed by human endometrial stromal cells during decidualization. However, the role for PTHLH in the decidualization process is unknown.

OBJECTIVE

To examine the effects of PTHLH on the induction and maintenance of decidualization of human uterine fibroblast (HUF) cells in vitro.

METHODS

HUF cells were treated with a PTHLH siRNA or a PTHLH receptor antagonist (bPTH(7-34)) before or after decidualization with medroxyprogesterone acetate (MPA), estradiol (E(2)), and prostaglandin E(2) (PGE(2)). Decidualization was monitored by immunocytochemistry and the induction of decidualization-specific marker genes, including IGFBP-1, prolactin, lefty, and transcription factor FOXO1.

RESULTS

HUF cells decidualized after pretreatment with a PTHLH siRNA showed greater morphologic changes of decidualization, greater IGFBP-1 protein, and two- to threefold more IGFBP-1, prolactin, lefty, and FOXO1 mRNAs than cells pretreated with a nonsilencing RNA. The PTHLH siRNA pretreated cells also had 31% less DNA fragmentation (TUNEL assay) and 30-35% less caspase 3 levels during decidualization than cells pretreated treated with nonsilencing RNA. Treatment of HUF cells with PTHLH siRNA or bPTH(7-34) at 9 d after the induction of decidualization also resulted in 2.1- to 3.2-fold greater IGFBP-1, prolactin, lefty, and FOXO1 mRNA levels than that noted in control cells treated with nonsilencing RNA.

CONCLUSIONS

These finding strongly suggest that PTHLH represses the induction of human decidualization, stimulates stromal cell apoptosis, and limits the extent of uterine stromal cell differentiation. Because PTHLH and its receptor are expressed by HUF cells and placental cells, the inhibitory effect of PTHLH on decidualization appears to be due, at least in part, to an autocrine/paracrine mechanism.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.

The study objective was to investigate the effects of fluoride on intact parathyroid hormone (iPTH) secretion. Thyro-parathyroid complexes (TPC) from C3H (n = 18) and B6 (n = 18) mice were cultured in Ca²⁺-optimized medium. TPC were treated with 0, 250, or 500 µM NaF for 24 h and secreted iPTH assayed by ELISA. C3H (n = 78) and B6 (n = 78) mice were gavaged once with distilled or fluoride (0.001 mg [F⁻]/g of body weight) water. At serial time points (0.5-96 h) serum iPTH, fluoride, total calcium, phosphorus, and magnesium levels were determined. Expression of genes involved in mineral regulation via the bone-parathyroid-kidney (BPK) axis, such as parathyroid hormone (Pth), calcium-sensing receptor (Casr), vitamin D receptor (Vdr), parathyroid hormone-like hormone (Pthlh), fibroblast growth factor 23 (Fgf23), α-Klotho (αKlotho), fibroblast growth factor receptor 1c (Fgf1rc), tumor necrosis factor 11 (Tnfs11), parathyroid hormone receptor 1 (Pth1r), solute carrier family 34 member 1 (Slc34a1), solute carrier 9 member 3 regulator 1 (Slc9a3r1), chloride channel 5 (Clcn5), and PDZ domain-containing 1 (Pdzk1), was determined in TPC, humeri, and kidneys at 24 h. An in vitro decrease in iPTH was seen in C3H and B6 TPC at 500 µM (p < 0.001). In vivo levels of serum fluoride peaked at 0.5 h in both C3H (p = 0.002) and B6 (p = 0.01). In C3H, iPTH decreased at 24 h (p < 0.0001), returning to baseline at 48 h. In B6, iPTH increased at 12 h (p < 0.001), returning to baseline at 24 h. Serum total calcium, phosphorus, and magnesium levels did not change significantly. Pth, Casr,αKlotho,Fgf1rc,Vdr, and Pthlh were significantly upregulated in C3H TPC compared to B6. In conclusion, the effects of fluoride on TPC in vitro were equivalent between the 2 mouse strains. However, fluoride demonstrated an early strain-dependent effect on iPTH secretion in vivo. Both strains demonstrated differences in the expression of genes involved in the BPK axis, suggesting a possible role in the physiologic handling of fluoride.

We have previously reported the expression of parathyroid hormone-like hormone (PTHLH) in well-differentiated, Schwannian stroma-rich neuroblastic tumors. The aim of this study was to functionally assess the role of PTHLH and its receptor, PTH1R, in neuroblastoma. Stable knockdown of PTHLH and PTH1R was conducted in neuroblastoma cell lines to investigate the succeeding phenotype induced both in vitro and in vivo. Downregulation of PTHLH reduced MYCN expression and subsequently induced cell cycle arrest, senescence, and migration and invasion impairment in a MYCN-amplified, TP53-mutated neuroblastoma cell line. These phenotypes were associated with reduced tumorigenicity in a murine model. We also show that PTHLH expression is not under the control of the calcium-sensing receptor in neuroblastoma. Conversely, its production is stimulated by epidermal growth factor receptor (EGFR). Accordingly, irreversible EGFR inhibition with canertinib abolished PTHLH expression. The oncogenic role of PTHLH appeared to be a consequence of its intracrine function, as downregulation of its receptor, PTH1R, increased anchorage-independent growth and induced a more undifferentiated, invasive phenotype. Respectively, high PTH1R mRNA expression was found in MYCN nonamplified primary tumors and also significantly associated with other prognostic factors of good outcome. This study provides the first evidence of the dual role of PTHLH in the behavior of neuroblastomas. Moreover, the identification of EGFR as a transcriptional regulator of PTHLH in neuroblastoma provides a novel therapeutic opportunity to promote a less aggressive tumor phenotype through irreversible inhibition of EGFR tyrosine kinase activity.

Dedifferentiated peripheral chondrosarcoma is a rare subtype of chondrosarcoma arising superimposed on the cartilage cap of a preexisting osteochondroma. It consists of two clearly defined components, a low-grade malignant, well-differentiated cartilage component and a high-grade non-cartilaginous sarcoma. Signaling pathways having a role in normal cartilage development were analyzed in these tumors, and compared with available data of other cartilaginous tumors. Sixteen well-characterized dedifferentiated peripheral chondrosarcomas were immunohistochemically analyzed for parathyroid hormone-like hormone (PTHLH)-BCL-2, fibroblastic growth factor (FGF), and transforming growth factor-beta signaling molecules, as well as matrix molecules and p53, comparing the chondrogenic component of dedifferentiated peripheral chondrosarcomas with the anaplastic component and with previously published data obtained from conventional grade I and II secondary peripheral chondrosarcomas. Results were correlated with clinical outcome. In the anaplastic component, various lines of differentiation could be found (collagen I (6/16), CD31 (1/16), smooth muscle actin (12/16), muscle-specific actin (12/16) and desmin (2/9)). Compared with the anaplastic component, the chondrogenic component of dedifferentiated peripheral chondrosarcomas shows more often expression of cyclin D1 (P=0.05), p53 (P=0.008), plasminogen activator inhibitor 1 (PAI-1) (P=0.005), and CD44 (P=0.030). Compared with secondary peripheral chondrosarcomas, more samples were positive in the chondrogenic component of dedifferentiated peripheral chondrosarcomas for FGF signaling (FGF receptor 3 P=0.000; bFGF P=0.003) and CD44 (P=0.000). Lower expression of BCL-2 (P=0.025) and absence of CD44v3 (P=0.000), a splice variant of CD44, was observed in the chondrogenic component of dedifferentiated peripheral chondrosarcomas compared with secondary peripheral chondrosarcomas. With regard to clinical data, PAI-1 expression in the chondrogenic component of dedifferentiated peripheral chondrosarcomas correlated with better survival (P=0.019). In conclusion, in the chondrogenic component of dedifferentiated peripheral chondrosarcomas, FGF signaling pathway is active, whereas PTHLH signaling seems to be low/downregulated. Interestingly, although the chondrogenic component of dedifferentiated peripheral chondrosarcoma is CD44+/CD44v3-, secondary peripheral chondrosarcomas is CD44-/CD44v3+, which suggest different splicing (preference). The prognostic value of PAI-1 in dedifferentiated peripheral chondrosarcomas might also be of interest for the more common dedifferentiated central chondrosarcomas.

The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric acid secretion was impaired and the ECL cell signaling pathway was inhibited in CCK2 receptor KO mice but not in CCK1 receptor KO mice. However, in CCK1+2 receptor double KO mice the acid secretion in response to pylorus ligation-induced vagal stimulation and the ECL cell pathway were partially normalized, which was associated with an up-regulated pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1). The basal part of the gastric mucosa expressed parathyroid hormone-like hormone (PTHLH) in a subpopulation of likely ECL cells (and possibly other cells) and vitamin D3 1α hydroxylase probably in trefoil peptide2-immunoreactive cells. In conclusion, mice lacking CCK receptors exhibited a functional shift from the gastrin-CCK pathways to the neuronal pathway in control of the ECL cells and eventually the acid secretion. Taking the present data together with previous findings, we suggest a possible link between gastric PTHLH and vitamin D and bone metabolism.