P311 Promotes Lung Fibrosis via Stimulation of Transforming Growth Factor-ß1, -ß2, and -ß3 Translation.

Interstitial lung fibrosis, a frequently idiopathic and fatal disease, has been linked to the increased expression of profibrotic transforming growth factor (TGF)-ßs. P311 is an RNA-binding protein that stimulates TGF-ß1, -ß2, and -ß3 translation in several cell types through its interaction with the eukaryotic translation initiation factor 3b. We report that P311 is switched on in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in the mouse model of bleomycin (BLM)-induced pulmonary fibrosis. To assess the in vivo role of P311 in lung fibrosis, BLM was instilled into the lungs of P311-knockout mice, in which fibrotic changes were significantly decreased in tandem with a reduction in TGF-ß1, -ß2, and -ß3 concentration/activity compared with BLM-treated wild-type mice. Complementing these findings, forced P311 expression increased TGF-ß concentration/activity in mouse and human lung fibroblasts, thereby leading to an activated phenotype with increased collagen production, as seen in IPF. Consistent with a specific effect of P311 on TGF-ß translation, TGF-ß1-, -ß2-, and -ß3-neutralizing antibodies downregulated P311-induced collagen production by lung fibroblasts. Furthermore, treatment of BLM-exposed P311 knockouts with recombinant TGF-ß1, -ß2, and -ß3 induced pulmonary fibrosis to a degree similar to that found in BLM-treated wild-type mice. These studies demonstrate the essential function of P311 in TGF-ß-mediated lung fibrosis. Targeting P311 could prove efficacious in ameliorating the severity of IPF while circumventing the development of autoimmune complications and toxicities associated with the use of global TGF-ß inhibitors.

The effect of combined pulsed wave low-level laser therapy and mesenchymal stem cell-conditioned medium on the healing of an infected wound with methicillin-resistant Staphylococcal aureus in diabetic rats.

This study aims to investigate the combined effects of Pulsed wave low-level laser therapy (PW LLLT) and human bone marrow mesenchymal stem cell-conditioned medium (hBM-MSC-CM) on the microbial flora and tensiometrical properties of an infected wound model with methicillin-resistant staphylococcal aureus (MRSA) in an experimental model for Type 1 diabetes mellitus (TIDM). TIDM was induced in rats by streptozotocin (STZ). One full-thickness excision was made on the backs of the rats. Next, the rats were divided into the following groups: Group 1 was the control (placebo) group; Group 2 received hBM-MSCs-CM four times; Group 3 were laser PWLLLT (890 nm, 80 Hz, 0.2 J/cm2 ); and Group 4 received hBM-MSCs-CM +LASER. Wounds were infected with MRSA. Microbiological examinations were performed on days 4, 7, and 15. Tensiometerical examinations were carried out on the 15th day. One-way analysis of variance showed that laser and CM alone and/or in combination significantly increases the tensiomerical properties of the repaired wounds compared with control wounds. A combination of PW laser and CM was statistically more effective than other treated groups. Two-way analysis of variance showed that laser and CM alone and/or in combination significantly decreases the colony-forming units (CFUs) compared with the control group. The application of hBM-MSC-CM and PWlaser alone and/or together significantly accelerates the wound-healing process in MRSA-infected cutaneous wounds in TI DM in rats. Additionally, a combined application of hBM-MSC-CM and PWlaser demonstrates a synergistic effect on the wound-healing process in MRSA-infected cutaneous wounds in Type I DM rats.

Neuronal Protein 3.1 Deficiency Leads to Reduced Cutaneous Scar Collagen Deposition and Tensile Strength due to Impaired Transforming Growth Factor-ß1 to -ß3 Translation.

Neuronal protein 3.1 (P311), a conserved RNA-binding protein, represents the first documented protein known to stimulate transforming growth factor (TGF)-ß1 to -ß3 translation in vitro and in vivo. Because TGF-ßs play critical roles in fibrogenesis, we initiated efforts to define the role of P311 in skin scar formation. Here, we show that P311 is up-regulated in skin wounds and in normal and hypertrophic scars. Genetic ablation of p311 resulted in a significant decrease in skin scar collagen deposition. Lentiviral transfer of P311 corrected the deficits, whereas down-regulation of P311 levels by lentiviral RNA interference reproduced the deficits seen in P311-/- mice. The decrease in collagen deposition resulted in scars with reduced stiffness but also reduced scar tensile strength. In vitro studies using murine and human dermal fibroblasts showed that P311 stimulated TGF-ß1 to -ß3 translation, a process that involved eukaryotic translation initiation factor 3 subunit b as a P311 binding partner. This resulted in increased TGF-ß levels/activity and increased collagen production. In addition, P311 induced dermal fibroblast activation and proliferation. Finally, exogenous TGF-ß1 to -ß3, each restituted the normal scar phenotype. These studies demonstrate that P311 is required for the production of normal cutaneous scars and place P311 immediately up-stream of TGF-ßs in the process of fibrogenesis. Conditions that decrease P311 levels could result in less tensile scars, which could potentially lead to higher incidence of dehiscence after surgery.

Evidence Supporting a Lymphatic Endothelium Origin for Angiomyolipoma, a TSC2(-) Tumor Related to Lymphangioleiomyomatosis.

Angiomyolipoma (AML) is a tumor closely related to lymphangioleiomyomatosis (LAM). Both entities are characterized by the proliferation of smooth muscle actin and melanocytic glycoprotein 100 (recognized by antibody HMB-45)-positive spindle-shaped and epithelioid cells. AML and LAM are etiologically linked to mutations in the tsc2 and tsc1 genes in the case of LAM. These genes encode the proteins tuberous sclerosis complex (TSC)-1 and TSC2, which are directly involved in suppressing the mechanistic target of rapamycin cell growth signaling pathway. Although significant progress has been made in characterizing and pharmacologically slowing the progression of AML and LAM with rapamycin, our understanding of their pathogenesis lacks an identified cell of origin. We used an AML-derived cell line to determine whether TSC2 restitution brings about the cell type from which AML arises. We found that AML cells express lymphatic endothelial cell markers consistent with lymphatic endothelial cell precursors in vivo and in vitro. Moreover, on TSC2 correction, AML cells mature into adult lymphatic endothelial cells and have functional attributes characteristic of this cell lineage, suggesting a lymphatic endothelial cell of origin for AML. These effects are dependent on TSC2-mediated mechanistic target of rapamycin inactivation. Finally, we demonstrate the in vitro effectiveness of norcantharidin, a lymphangiogenesis inhibitor, as a potential co-adjuvant therapy in the treatment of AML.

Novel RNA-binding protein P311 binds eukaryotic translation initiation factor 3 subunit b (eIF3b) to promote translation of transforming growth factor ß1-3 (TGF-ß1-3).

P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-ß1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 µm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-ß1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-ß 5'UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-ßs 5'UTRs and eIF3b, stimulates the translation of TGF-ß1, 2, and 3.

TIPs are tension-responsive proteins involved in myogenic versus adipogenic differentiation.

Stretch induces lung embryonic mesenchymal cells to follow a myogenic pathway. Using this system we identified a set of stretch-responsive factors, which we referred to as TIPs (tension-induced/inhibited proteins). TIPs displayed signature motifs characteristic of nuclear receptor coregulators and chromatin remodeling enzymes. A genomic BLAST search suggested that the three TIPs identified were isoforms originated by alternative splicing from a single gene. Functional studies revealed that TIP-1 and TIP-3 were involved in the cell's selection of the myogenic or the adipogenic pathway. TIP-1, induced by stretch, promoted myogenesis, while TIP-3, inhibited by stretch, stimulated adipogenesis. The selection involved TIP-mediated chromatin remodeling via a histone acetylation process and depended on TIP-1 and TIP-3 nuclear receptor binding boxes (NRBs). This study, therefore, suggests a new developmental mechanism linking the presence or absence of tension with divergent differentiation pathways.

Variability in protein cargo detection in technical and biological replicates of exosome-enriched extracellular vesicles.

Exosomes are extracellular vesicles (EVs) of ~20-200 nm diameter that shuttle DNAs, RNAs, proteins and other biomolecules between cells. The large number of biomolecules present in exosomes demands the frequent use of high-throughput analysis. This, in turn, requires technical replicates (TRs), and biological replicates (BRs) to produce accurate results. As the number and abundance of identified biomolecules varies between replicates (Rs), establishing the replicate variability predicted for the event under study is essential in determining the number of Rs required. Although there have been few reports of replicate variability in high throughput biological data, none of them focused on exosomes. Herein, we determined the replicate variability in protein profiles found in exosomes released from 3 lung adenocarcinoma cell lines, H1993, A549 and H1975. Since exosome isolates are invariably contaminated by a small percentage of ~200-300 nm microvesicles, we refer to our samples as exosome-enriched EVs (EE-EVs). We generated BRs of EE-EVs from each cell line, and divided each group into 3 TRs. All Rs were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS) and customized bioinformatics and biostatistical workflows (raw data available via ProteomeXchange: PXD012798). We found that the variability among TRs as well as BRs, was largely qualitative (protein present or absent) and higher among BRs. By contrast, the quantitative (protein abundance) variability was low, save for the H1975 cell line where the quantitative variability was significant. Importantly, our replicate strategy identified 90% of the most abundant proteins, thereby establishing the utility of our approach.

High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis.

Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621-2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027-3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321-1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341-1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase-PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.

The effect of combined photobiomodulation and curcumin on skin wound healing in type I diabetes in rats.

The purpose of the present scientific study was to analyze the effects of combined pulsed wave Photobiomodulation (PW PBM) and Curcumin on the microbial flora; in addition, the tensiometrical wounds properties for type one diabetes mellitus (TIDM) in an experimental animal model. TIDM induction was performed in thirty rats. In the entire animals, one full-thickness excision was implemented on their backs. Randomly, the divisions of rats into 5 groups took place. The primary group was considered as the control group and did not receive any treatment. The secondary group (placebo) received sesame oil by gastric gavage. The third group received PWPBM (890 nm, 80 Hz, 0.2 J/cm2). The fourth group received curcumin (40 mg/kg, which was dissolved in sesame oil) by gastric gavage. Eventually, the fifth group received PW PBM + curcumin. Precisely, on day 7, microbiological examinations, and on the 15th day microbiological and tensiometrical examinations were conducted. The data were analyzed by statistical tests. PW PBM, significantly exacerbated tensiometrical properties of the TIDM repairing wound. PW PBM, curcumin, and PWPBM + curcumin significantly decreased colony forming units compared to the control and the placebo groups indeed. It was remarkably attained that PW PBM significantly accelerated the process of wound healing in the STZ-induced TIDM. The PW PBM was statistically more compelling compared to the curcumin and PWPBM + curcumin. PW PBM, curcumin, and PWPBM + curcumin significantly decreased colony forming units compared to the control and placebo groups.

P311 induces a TGF-beta1-independent, nonfibrogenic myofibroblast phenotype.

P311, also called PTZ17, was identified by suppressive subtraction hybridization as potentially involved in smooth muscle (SM) myogenesis. P311 is an 8-kDa protein with several PEST-like motifs found in neurons and muscle. P311 transfection into two fibroblast cell lines, NIH 3T3 and C3H10 T1/2, induced phenotypic changes consistent with myofibroblast transformation, including upregulation of SM alpha-actin and SM22, induction of FGF-2, VEGF, PDGF, and PDGF receptors, upregulation of integrins alpha3 and alpha5, and increased proliferation rate. The P311-mediated changes differed, however, from the well-characterized myofibroblast in that P311 inhibited TGF-beta1, TGF-beta receptor 2, and TGF-beta1-activating MMP-2 and MMP-9, with the resultant decrease in collagen 1 and 3 expression. The effect of P311 on collagen was overcome by exogenous TGF-beta1, indicating that the cells were responsive to TGF-beta1 paracrine stimulus. In support of a role for P311 in vivo, immunohistochemical examination of human wounds showed P311 only in myofibroblasts and their activated precursors. To our knowledge, these studies are the first to implicate P311 in myofibroblast transformation, to demonstrate that transformation may occur independently of TGF-beta1, and to suggest that P311 may prevent fibrosis.

A potential oncogenic activity of platelet-derived growth factor d in prostate cancer progression.

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH(2)-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the beta-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer.

Role of stretch in activation of smooth muscle cell lineage.

Mechanical force is a critical modulator of smooth muscle (SM) function and gene expression. Very little is known, however, about its contribution to SM myogenesis. This review presents and discusses what has been learned about the role of mechanical force in inducing SM myogenesis and some of the signaling mechanisms involved in this process.

Reduced fibroblast interaction with intact collagen as a mechanism for depressed collagen synthesis in photodamaged skin.

This report provides evidence from a number of different approaches (i.e., comparison of cell shape in 1-microm sections of photodamaged versus healthy skin at the light microscopic level; comparison of cell shape and apposition to collagen fibrils in ultrathin sections of the same tissues examined by transmission electron microscopy, and fluorescence staining for adhesion site protein expression and actin filament architecture in frozen tissue sections) that dermal cells in healthy skin are attached to collagen fibrils over a large part of the cell border, have a flattened/spread (two-dimensional) appearance and have abundant actin in their cytoplasm. In contrast, cells in photodamaged skin are often in contact with fragmented collagen or amorphous debris rather than intact collagen, have a collapsed/elongated shape, and have a lower amount of actin. Collagen synthesis is reduced in severely photodamaged skin relative to collagen synthesis in corresponding sun-protected skin (N Engl J Med 329:530, 1993). We hypothesize that fibroblasts in severely damaged skin have less interaction with intact collagen and as a result experience a reduction in mechanical tension. Decreased collagen synthesis is (presumed to be) the result.

Combined smooth muscle and melanocytic differentiation in lymphangioleiomyomatosis.

Pulmonary lymphangioleiomyomatosis (LAM) is characterized by abnormal proliferation of immature-looking smooth muscle (SM)-like cells (LAM cells), leading to lung destruction and cyst formation. In addition to expressing some SM markers, scattered LAM cells express the melanocytic maker gp100, which is recognized by antibody HMB45, suggesting that at least a few LAM cells may have melanocytic differentiation. Here we immunostained 26 LAM samples for several melanocyte-related proteins. These studies showed that all LAM cells express tetraspanin CD63, a melanoma-associated protein that belongs to the transmembrane 4 superfamily. The majority of LAM cells also immunoreacted with PNL2, an antibody against a yet uncharacterized melanocytic antigen. Furthermore, we examined the co-expression of PNL2 and Ki-67, an indicator of cell proliferation, and found that PNL2-positive LAM cells showed a significantly lower proliferation rate compared with their negative counterparts. Our findings shed new light on the nature of the LAM cells by demonstrating their combined SM and melanocytic differentiation and the existence of subpopulations with different proliferative potential. Furthermore, these studies provided two new antibodies useful in the diagnosis of LAM.

In pulmonary lymphangioleiomyomatosis expression of progesterone receptor is frequently higher than that of estrogen receptor.

Lymphangioleiomyomatosis (LAM) of the lung is a rare low-grade malignancy affecting primarily women of childbearing age. LAM is characterized by the proliferation of SMA and HMB-45 positive spindle-shaped and epithelioid cells throughout the lung in the form of discrete lesions causing cystic destruction and ultimately respiratory insufficiency. LAM occurs sporadically or in patients with tuberous sclerosis complex (TSC) and is etiologically linked to mutations in the TSC1 and TSC2 genes. Although LAM cells are known to express estrogen and progesterone receptors (ER and PR, respectively), their respective expression level was never determined. Therefore, here we measured the immunohistochemical expression of ERs and PRs in a large series of pulmonary LAM cases using the Aperio Spectrum Analysis Platform. Our case series comprised open lung biopsy specimens from 20 LAM patients and lungs explanted during the course of lung transplant from 24 patients. All cases were positive for ER and PR. PR expression was statistically significantly higher than ER in 80 % of the biopsies while ER predominated only in one case. Specimens from explanted cases of LAM had relatively fewer PR-positive nuclei. As a result, PR expression was significantly higher than ER in 38 % of the cases, whereas ER predominated in 33 %. Overall, PR expression predominated in 57 % of cases and ER in 21 %. These data indicate that PR frequently prevails over ER in pulmonary LAM. LAM is unusual in its high PR/ER ratio; other female neoplasms show a definite prevalence of ER. Our findings therefore warrant further study of PR function in LAM.

Lymphatic endothelial differentiation in pulmonary lymphangioleiomyomatosis cells.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare, low-grade neoplasm affecting almost exclusively women of childbearing age. LAM belongs to the family of perivascular epithelioid cell tumors, characterized by spindle and epithelioid cells with smooth muscle and melanocytic differentiation. LAM cells infiltrate the lungs, producing multiple, bilateral lesions rich in lymphatic channels and forming cysts, leading to respiratory insufficiency. Here we used antibodies against four lymphatic endothelial markers-podoplanin (detected by D2-40), prospero homeobox 1 (PROX1), vascular endothelial growth factor receptor 3 (VEGFR-3), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-to determine whether LAM cells show lymphatic differentiation. Twelve of 12 diagnostic biopsy specimens (early-stage LAM) and 19 of 19 explants (late-stage LAM) showed immunopositivity for D2-40 in most neoplastic cells. PROX1, VEGFR-3, and LYVE1 immunoreactivity varied from scarce in the early stage to abundant in the late stage. Lymphatic endothelial, smooth muscle, and melanocytic markers were partially co-localized. These findings indicate that lymphatic endothelial differentiation is a feature of LAM and provide evidence of a previously unidentified third lineage of differentiation in this neoplasm. This study has implications for the histological diagnosis of LAM, the origin of the neoplastic cells, and potential future treatment with drugs targeting lymphangiogenesis.

Blood pressure homeostasis is maintained by a P311-TGF-ß axis.

P311 is an 8-kDa intracellular protein that is highly conserved across species and is expressed in the nervous system as well as in vascular and visceral smooth muscle cells. P311-null (P311-/-) mice display learning and memory defects, but alterations in their vasculature have not been previously described. Here we report that P311-/- mice are markedly hypotensive with accompanying defects in vascular tone and VSMC contractility. Functional abnormalities in P311-/- mice resulted from decreased total and active levels of TGF-ß1, TGF-ß2, and TGF-ß3 that arise as a specific consequence of decreased translation. Vascular hypofunctionality was fully rescued in vitro and in vivo by exogenous TGF-ß1-TGF-ß3. Conversely, P311-transgenic (P311(TG)) mice had elevated levels of TGF-ß1-TGF-ß3 and subsequent hypertension. Consistent with findings attained in mouse models, arteries recovered from hypertensive human patients displayed increased P311 expression. Thus, we identified P311 as the first protein known to modulate TGF-ß translation and the first pan-regulator of TGF-ß expression under steady-state conditions. Together, our findings point to P311 as a critical blood pressure regulator and establish a potential link between P311 expression and the development of hypertensive disease.

Embryological origin of airway smooth muscle.

Airway smooth muscle (SM) develops from local mesenchymal cells located around the tips of growing epithelial buds. These cells gradually displace from distal to proximal position alongside the bronchial tree, elongate, and begin to synthesize SM-specific proteins. Mechanical tension (either generated by cell spreading/elongation or stretch), as well as epithelial paracrine factors, regulates the process of bronchial myogenesis. The specific roles of many of these paracrine factors during normal lung development are currently unknown. It is also unknown how and if mechanical and paracrine signals integrate into a common myogenic pathway. Furthermore, as with vascular SM and other types of visceral SM, we are just beginning to elucidate the intracellular signaling pathways and the genetic program that controls lung myogenesis. Here we present what we have learned so far about the embryogenesis of bronchial muscle.

Effects of the SANT domain of tension-induced/inhibited proteins (TIPs), novel partners of the histone acetyltransferase p300, on p300 activity and TIP-6-induced adipogenesis.

We previously identified a set of transcription regulators, referred to as TIPs (tension-induced/inhibited proteins), with a role in myogenic versus adipogenic differentiation. Here we report that the TIP family comprises eight isoforms, all bearing a SANT (switching-defective protein 3, adaptor 2, nuclear receptor corepressor, and transcription factor IIIB) domain and some of them presenting S-adenosyl-l-methionine (SAM) and nuclear receptor box (NRB) motifs, all characteristic of histone-modifying enzymatic complexes. TIPs have SANT-dependent, p300-mediated histone acetyltransferase (HAT) activity. Ectopic TIP-6 (SANT(+) SAM(-) NRB(-)) but not TIP-6DeltaSANT induced de novo PPARgamma2-mediated adipogenic gene expression in NIH 3T3 cells and promoted preadipocyte differentiation into fat cells. TIP-6 was also involved in mediating hormonally/biochemically induced adipogenic differentiation of 3T3-L1 cells. Furthermore, TIP-6 was identified in adipose tissue in vivo. TIP-6 bound directly and indirectly to p300 and histone H4 (H4). Deletion of the SANT domain did not abolish TIP-6 interaction with p300 and H4 but eliminated direct TIP-6 binding to p300. Chromatin immunoprecipitation assays showed the recruitment of TIP-6, TIP-6DeltaSANT, and p300 to the PPARgamma2 promoter, but H3/H4 acetylation occurred only when p300 was directly associated with TIP-6. These studies demonstrated the importance of TIPs in the recruitment of p300 to specific promoters and in the regulation of p300 HAT activity through the involvement of the SANT domain. Furthermore, we identified TIP-6 as a new member of the adipogenic cascade.