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Cdk9, previously known as PITALRE, belongs to the Cdc2 family of protein kinases. We report the isolation and characterization of the complete gene coding for the murine Cdk9 protein. The gene contains seven exons spanning over 6 kb of genomic DNA, and the exon/intron boundaries conformed to the GT/AG rule. The Cdk9 gene mapped on mouse chromosome 2, which is consistent with the known region of synteny with human chromosome 9q34.1. The length of the individual exons ranged from 82 to 850 bp, and introns ranged from 452 to 1,465 bp. The further 5' flanking region of the gene showed features of a housekeeping promoter, such as the lack of a canonical TATA box and the presence of a CCAAT box as well as several GC boxes, which are potential binding sites for numerous transcription factors. Additionally, we performed a basic analysis of the transcriptional activity of the promoter and found that the 364 bp of Cdk9 5' flanking region were able to elicit high transcriptional levels of a luciferase reporter gene in NIH3T3 cells. This study provides the molecular basis for understanding the transcriptional control of the Cdk9 gene, and could serve to facilitate the molecular genetic investigation of Cdk9 function during mouse embryonal development.

To evaluate transcriptional mechanisms during cytokine induction of myeloid progenitor cell proliferation, we examined the expression and activity of transcription factors that control cell cycle-dependent histone genes in interleukin-3 (IL-3)-dependent FDC-P1 cells. Histone genes are transcriptionally upregulated in response to a series of cellular regulatory signals that mediate competency for cell cycle progression at the G1/S-phase transition. We therefore focused on factors that are functionally related to activity of the principal cell cycle progression at the G1/S-phase transition. We therefore focused on factors that are functionally related to activity of the principal cell cycle regulatory element of the histone H4 promoter:CDC2, cyclin A, as well as RB-and IRF-related proteins. Comparisons were made with activities of ubiquitous transcription factors that influence a broad spectrum of promoters independent of proliferation or expression of tissue-specific phenotypic properties. Northern blot analysis indicates that cellular levels of cyclin A and CDC2 mRNAs increase when DNA synthesis and H4 gene expression are initiated, supporting invoulvement in cell cycle progression. Using gel-shift assays, incorporating factor-specific antibody and oligonucleotide competition controls, we define three sequential periods following cytokine stimulation of FDC-P1 cells when selective upregulation of a subset of transcription factors is observed. In the initial period, the levels of SP1 and HiNF-P are moderately elevated; ATF, AP-1, and HiNF-M/IRF-2 are maximal during the second period; while E2F and HiNF-D, which contain cyclin A as a component, predominate during the third period, coinciding with maximal H4 gene expression and DNA synthesis. Differential regulation of H4 gene transcription factors following growth stimulation is consistent with a principal role of histone gene promoter elements in integrating cues from multiple signaling pathways that control cell cycle induction and progression. Regulation of transcription factors controlling histone gene promoter activity within the context of a staged cascade of responsiveness to cyclins and other physiological mediators of proliferation in FDC-P1 cells provides a paradigm for experimentally addressing interdependent cell cycle and cell growth parameters that are operative in hematopoietic stem cells. © 1995 Wiley-Liss, Inc.

The Rb2/p130 protein has been shown to have a high sequence homology with the retinoblastoma gene product (pRb), one of the most well‐characterized tumor suppressor genes, and with pRb‐related p107, especially in their conserved pocket domains, which display a primary role in the function of these proteins. In this study, we report on the biochemical and immunocytochemical characterization of the Rb2/p130 protein, using a polyclonal antibody developed against its “spacer” region included in the pocket domain of the whole protein. We show that pRb/p130 is a phosphoprotein located at the nuclear level and that its phosphorylation pathway can be dramatically reduced by phosphatase treatment. Moreover pRb/p130, with p107, with p107, is one of the major targets of the E1A viral oncoprotein‐associated kinase activity, showing a phosphorylation pattern which is modulated during the cell cycle, reaching a peak of activation at the onset of S‐phase. © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc.

It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse a- and b-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states. J. Cell. Biochem. 77:529-539, 2000. © 2000 Wiley-Liss, Inc.

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferen- tially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages. J. Cell. Biochem. 75:698-709, 1999. r 1999 Wiley-Liss, Inc.

We investigate the role played by dendritic cells (DCs) in the non-obese diabetic (NOD) mouse pancreas. The early peri-islet, nondestructive infiltration phase, and intra-islet, destructive infiltration phase, which immediately precedes overt diabetes, are studied. Results show that infiltrating cells are Ia-b, ICAM-1, and, mainly, MIDC-8 immunoreactive (ir). These data from silica-treated animals and ultrastructural observations strongly support the hypothesis that DCs are both Ia-b-ir and ICAM-1-ir and that they exert a pivotal role during the period of early infiltration. This is a novel finding for NOD mice and increases the interest for this protective cell type during the rather complex islet infiltration process. Moreover, the cytokine profile demonstrates that Th2 protective cytokines are specific for peri-islet infiltrate. Disappearance of DCs from the infiltrate is concomitant with both the formation of intra-islet infiltration and the increase in proinflammatory Th1 cytokine levels. This further supports the hypothesis that DCs may exert a protective role against diabetes development.

Neuroblastoma cells can undergo neural differentiation upon treatment with a variety of chemical inducers and growth factors. During this process, many cell cycle-related genes are downregulated while differentiation-specific genes are triggered. The retinoblastoma family proteins, pRb, p107, and pRb2/p130, are involved in transcriptional repression of proliferation genes, mainly through their interaction with the E2F transcription factors. We report that pRb2/p130 expression levels increased during differentiation of neuroblastoma cell line LAN-5. On the other hand, both pRb and p107 decreased and underwent progressive dephosphorylation at late differentiation times. The expression of B-myb and c-myb, two targets of the retinoblastoma family proteins, were downregulated in association with the increase of pRb2/p130, which was detected as the major component of the complex with E2F on the E2F site of the B-myb promoter in differentiated cells. Interestingly, E2F4, a preferential partner of p107 and pRb2/p130, was upregulated and underwent changes in cellular localization during differentiation. In conclusion, our data suggest a major role of pRb2/p130 in the regulation of B-myb promoter during neural differentiation despite the importance of cofactors in modulating the function of the retinoblastoma family proteins.

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase, and the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the homeodomain CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, we have investigated these determinants of transcriptional regulation at the G1/S phase transition in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. We find striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. We also observe a dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal. J. Cell. Biochem. 66:512–523, 1997. © 1997 Wiley-Liss, Inc.