CD11c as a transcriptional biomarker to predict response to anti-TNF monotherapy with adalimumab in patients with rheumatoid arthritis.

We performed transcription profiling using monocytes to identify predictive markers of response to anti-tumor necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA). Several potential predictors of response were identified, including CD11c. Validation in samples from independent cohorts (total of n = 27 patients) using reverse transcription-PCR confirmed increased expression of CD11c in responders to adalimumab (100% sensitivity; 91.7% specificity, power 99.6%; alpha = 0.01). Pretherapy CD11c levels significantly correlated with the response criteria as defined by the American College of Rheumatology (ACR) (r = 0.656, P < 0.0001). However, CD11c was neither predictive of response to methotrexate (MTX) alone (n = 34) nor to MTX in combination with adalimumab (n = 16). Clinical responders revealed a reset to a normal expression pattern of resident/inflammatory monocyte markers, which was absent in nonresponders. Therefore, an analysis of key cell types identifies potentially predictive biomarkers that may help to restrict the use of adalimumab to therapy responders. Larger studies, including studies of monotherapy with other drugs, are now needed to confirm and validate the specificity of CD11c for anti-TNF biologics.

An alternative Pit-1 RNA splicing product reveals modular binding and nonmodular transcriptional activities of the POU-specific domain.

We have identified a form of the pituitary-specific POU protein Pit-1 that results from deletion of the POU-specific (POUs) domain by alternative RNA splicing. This natural variant of Pit-1 (called delta 4Pit-1) has revealed several aspects of the function of the POUs domain. The delta 4Pit-1 protein was characterized using a delta 4Pit-1-specific antiserum. Further, selection assays of random oligonucleotide pools identified binding site preferences for both wild type and delta 4Pit-1. Methylation interference, copper phenanthrolene, and missing contact analyses were used to compare the binding characteristics of the two forms of Pit-1 on a selected site. DNA binding affinity assays on several DNA elements revealed that the POUs domain contains a modular DNA binding activity affecting the DNA binding affinity of the entire POU domain on some, but not on other, DNA sites. Functional analysis on such DNA elements has revealed that the POUs domain is an essential, but nonmodular, component of the Pit-1 trans-activation domain dependent on its natural context within the Pit-1 protein.

Immunohistochemical expression of PIT-1 protein in pituitary glands of human GRF transgenic mice: its relationship with hormonal expressions.

It has been suggested that pit-1 protein may play a role in the differentiation of the anterior pituitary cells. The present immunohistochemical studies were designed to elucidate the relationship between functional differentiation of pituitary adenoma and expression of pit-1 protein in human (h) GRF transgenic mice. Pituitaries from a 10 month old and a 6 month old transgenic mice were fixed in 4% paraformaldehyde and embedded in paraffin. The indirect immunoperoxidase method was performed using antibodies against hGRF, GH, PRL, ACTH, alpha subunit (SU), FSH beta SU, LH beta SU, TSH beta SU, and pit-1 protein. Immunohistochemical double staining was performed at light and electron microscopic levels. The pituitary glands of hGRF transgenic mice (both 10 month and 6 month old) demonstrated diffuse hyperplasia of GH positive cells with coexpression of hGRF within the same cells. There were also scattered cells which were positive for other hormones and hormone subunits in the hyperplastic pituitary. Three discrete nodules were found in the pituitary gland of a 10 month old hGRF transgenic mouse and were identified as adenomas. These adenomas were composed of enlarged round cells which were positive only for GH, hGRF, PRL and TSH beta SU. Pit-1 protein was intensely expressed in the nuclei of the adenoma cells. These results suggest the existence of an autocrine mechanism by hGRF in the formation of somato-lacto-thyrotroph adenoma via constitutive pit-1 expression.

POU-domain proteins Pit-1 and Oct-1 interact to form a heteromeric complex and can cooperate to induce expression of the prolactin promoter.

Two members of the POU-domain family of transcriptional activators, Pit-1 and Oct-1, are coexpressed in cells of the anterior pituitary gland. We demonstrate that the pituitary-specific developmental regulator Pit-1 can bind as a heterodimer with the widely expressed 100-kD Oct-1 protein to critical tissue-specific cis-active elements in the rat prolactin gene. Pit-1 and Oct-1 can also associate, in the absence of DNA, via their POU domains. Coexpression of Pit-1 and Oct-1 in a heterologous cell type results in reproducible 2- to 2.5-fold synergistic transcriptional effects on genes under control of the native prolactin promoter or of a single Pit-1 response element. These results suggest that a combinatorial pattern of heterodimeric and homodimeric interactions between different members of the POU-domain gene family can result when members of this large family are coexpressed in cells of developing and established organ systems, potentially regulating differential developmental gene activation.

Alternative translation initiation site usage results in two structurally distinct forms of Pit-1.

Pit-1 is a pituitary-specific transcription factor that plays a critical role in the normal development of the anterior pituitary gland. Previous analyses have shown that this protein exists in the rat pituitary gland and in rat pituitary-derived cell lines as two forms of relative molecular mass 33 and 31 kDa. This aspect of Pit-1 expression has been conserved throughout the evolution from rodents to humans. Here, we determine the origin of these structurally distinct forms of Pit-1 protein and find that these arise as a consequence of the alternative usage of translation initiation sites present in Pit-1 mRNA.

The POU-specific domain of Pit-1 is essential for sequence-specific, high affinity DNA binding and DNA-dependent Pit-1-Pit-1 interactions.

Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more divergent homeodomain (POUHD). Analysis of mutant Pit-1 proteins suggests that, while the POUHD is required and sufficient for low affinity DNA binding, the POUS domain is necessary for high affinity binding and accurate recognition of natural Pit-1 response elements. Pit-1 is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNA-dependent protein-protein interactions requiring the POUS domain. Analysis of alpha-helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUS domain contacting DNA. Transcriptional activity of Pit-1 on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs.

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-1 protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-1, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-1, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.