Human Preosteoblastic Cell Culture from a Patient with Severe Tumoral Calcinosis-Hyperphosphatemia Due to a New GALNT3 Gene Mutation: Study of In Vitro Mineralization.

Human disorders of phosphate (Pi) handling and skeletal mineralization represent a group of rare bone diseases. One of these disease is tumoral calcinosis (TC). In this study, we present the case of a patient with TC with a new GALNT3 gene mutation. We also performed functional studies using an in vitro cellular model. Genomic DNA was extracted from peripheral blood collected from a teenage Caucasian girl affected by TC, and from her parents. A higher capability to form mineralization nodules in vitro was found in human preosteoblastic cells of mutant when compared to wild-type controls. We found a novel homozygous inactivating splice site mutation in intron I (c.516-2a>g). A higher capability to form mineralization nodules in vitro was found in the mutant cells in human preosteoblastic cells when compared to wild-type controls. Understanding the functional significance and molecular physiology of this novel mutation will help to define the role of FGF23 in the control of Pi homeostasis in normal and in pathological conditions.

Variable expression of the transcription factors cAMP response element-binding protein and inducible cAMP early repressor in the normal adrenal cortex and in adrenocortical adenomas and carcinomas.

The molecular mechanisms leading to adrenocortical tumorigenesis have been only partially elucidated so far. Because the pituitary hormone ACTH, via activation of the cAMP pathway, regulates both cell proliferation/differentiation and steroid synthesis in the adrenal cortex, in this study we focused on the cAMP-dependent transcription factors cAMP responsive element modulator (CREM) and cAMP responsive element binding protein (CREB). We studied CREM and CREB expression by RT-PCR in human normal adrenal cortex (n = 3), adrenocortical adenomas (n = 8), and carcinomas (n = 8). We found transcripts corresponding to the isoforms alpha, beta, gamma, and tau2 of the CREM gene in all of the normal adrenal tissues, in the adenomas, and in seven of eight carcinomas. On the other hand, mRNA for the inducible cAMP early repressor isoforms, which derive from an internal promoter of CREM gene, was detected in the normal adrenal and in seven of eight adenomas, but in only three of eight carcinomas. Similarly, CREB transcripts were readily detectable in all normal adrenals and adenomas, whereas they were not found in four of eight adrenal carcinomas. To further characterize the carcinomas, telomerase activity and the expression of the ACTH receptor gene were determined. Telomerase activity in the carcinomas resulted in levels significantly higher than in the adenomas, whereas the levels of ACTH receptor mRNA were lower in the carcinomas. No correlation was found in the carcinomas between the levels of the ACTH receptor transcript and the loss of expression of CREB/inducible cAMP early repressor, suggesting that this alteration is not secondary to an upstream disregulation at the receptor level. In conclusion, our results suggest that an alteration in cAMP signaling may be associated with malignancies of the adrenal cortex.

Expression of cyclic adenosine 3',5'-monophosphate (cAMP)-responsive element binding protein and inducible-cAMP early repressor genes in growth hormone-secreting pituitary adenomas with or without mutations of the Gsalpha gene.

In about 30-40% of GH-secreting adenomas, gain-of-function mutations of the Gsalpha gene, which convert this gene into an oncogene termed gsp, occur. Gsalpha mutations have been related to pituitary tumorigenesis. We focused on 2 nuclear transcription factors that are final targets of the cAMP-dependent pathway and are positively regulated by cAMP signaling, i.e. the cAMP-responsive element binding protein (CREB) and the inducible cAMP early repressor (ICER), that derives from alternative splicing of cAMP-responsive element modulator gene. We examined 21 GH-secreting adenomas, 8 with (gsp(+)) and 13 without (gsp(-)) a mutated Gsalpha. Analysis of CREB and ICER I/II messenger RNA revealed that the levels of both transcripts were higher in gsp(+) than in gsp(-) tumors (CREB/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mean optical density +/- SE, 2.34 +/- 0.36 in gsp(+) vs. 0.99 +/- 0.22 in gsp(-), P = 0.003; ICER I/GAPDH, 0.53 +/- 0.15 in gsp(+) vs. 0.14 +/- 0.07 in gsp(-), P = 0.01; ICER II/GAPDH, 1.5 +/- 0.21 in gsp(+) vs. 0.83 +/- 0.13 in gsp(-), P = 0.01), although a few cases in both groups did not display this pattern of expression. Moreover, no positive correlation between the levels of CREB and ICER transcripts was observed, suggesting the possible presence of alterations in the mechanisms by which cAMP signaling directs the expression of CREB and/or ICER genes. Our results indicate a complex pattern of expression of nuclear transcription factors that mediate cAMP action in both gsp(+) and gsp(-) tumors, suggesting that, beside Gsalpha gene mutations, different and partially unknown molecular events may contribute to the pathogenesis of these tumors.

Uteroglobin reverts the transformed phenotype in the endometrial adenocarcinoma cell line HEC-1A by disrupting the metabolic pathways generating platelet-activating factor.

Uteroglobin, originally named blastokinin, is a protein synthesized and secreted by most epithelia, including the endometrium. Uteroglobin has strong anti-inflammatory properties that appear to be due, at least in part, to its inhibitory effect on the activity of the enzyme phospholipase A(2). In addition, recent experimental evidence indicates that uteroglobin exerts antiproliferative and antimetastatic effects in different cancer cells via a membrane receptor. The human endometrial adenocarcinoma cell line HEC-1A does not express uteroglobin. Thus, we transfected HEC-1A cells with human uteroglobin cDNA. The transfectants showed a markedly reduced proliferative potential as assessed by impaired plating efficiency as well as by reduced growth in soft agar. Cytofluorimetric analysis clearly indicated that in uteroglobin-transfected cells the time for completion of the cell cycle was increased. We previously demonstrated that HEC-1A cells actively synthesize platelet-activating factor, one of the products of phospholipase A(2) activity. In addition, we demonstrated that platelet-activating factor stimulates the proliferation of these cells through an autocrine loop. In uteroglobin transfectants, the activity of phospholipase A(2) and platelet-activating factor acetyl-transferase, which are involved in the synthesis of platelet-activating factor, was significantly reduced compared with wild-type and vector-transfected cells (p < 0.05). Our results indicate that enforced expression of uteroglobin in HEC-1A cells markedly reduced their growth potential and significantly impaired the synthesis of platelet-activating factor, an autocrine growth factor for these cells. These data suggest that one possible mechanism for the recently observed antineoplastic properties of uteroglobin may be the inhibition of the synthesis of platelet-activating factor.

Cyclic adenosine 3',5'-monophosphate-responsive element modulator gene expression in germ cells of normo- and oligoazoospermic men.

In about one third of infertile men the cause of impaired spermatogenesis is not known. Spermatogenesis appears to be mediated at least in part by the pituitary gonadotropins, which activate the cAMP-dependent signaling pathway. The end point of this pathway is the activation of nuclear transcription factors, such as cAMP-responsive element-binding protein and cAMP-responsive element modulator (CREM). These factors, upon binding to gene sequences identified as cAMP response elements, modulate the expression of germ cell-specific genes that, in turn, promote the completion of spermatogenesis. The expressions of the cAMP-responsive element-binding protein and CREM genes create different isoforms, which can be divided into two groups: activators or repressors of gene regulation. Only CREM repressors are expressed in premeiotic germ cells in mice, whereas a switch to the expression of the CREM activator tau is observed from postmeiotic germ cells onward. Completion of germ cell maturation appears to be dependent on this phenomenon. Recently, mice lacking CREM gene expression have been generated. These animals were infertile and presented a developmental arrest of germ cell maturation at the stage of early spermatid. In this report we demonstrate that CREM gene expression also occurs in human germ cells. In particular, we determined by RT-PCR that a switch from the expression of CREM repressors to CREM activators is present in postmeiotic germ cells in normospermic men. Conversely, in oligoazoospermic patients only the expression of CREM repressors was detected. These data were confirmed by in situ hybridization studies in which transcripts for CREM activators were detected in postmeiotic germ cells in testis specimens showing conserved spermatogenesis, but not in specimens showing maturation arrest at the spermatid stage. Thus, our results indicate that the lack of a switch in the expression of CREM gene isoforms may be related to impaired spermatogenesis in humans.