The TGF-ß/HDAC7 axis suppresses TCA cycle metabolism in renal cancer.

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1α, which is also lost in RCC tissues. PGC-1α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-ß signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF-ß restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF-ß/HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

PGC1α suppresses kidney cancer progression by inhibiting collagen-induced SNAIL expression.

The transcriptional events that promote invasive and metastatic phenotypes in renal cell carcinoma (RCC) remain poorly understood. Here we report that the decreased expression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α) and the increased expression of several genes encoding collagen family members are associated with RCC tumor progression. PGC1α restoration attenuates invasive phenotypes and suppresses tumor progression in vivo. In contrast, collagens produced by RCC cells promote invasive and migratory phenotypes. PGC1α restoration suppresses the expression of collagens and tumor phenotypes via the induction of miR-29a. Furthermore, decreased collagens via the PGC1α/miR-29a axis suppresses collagen-mediated activation of discoidin domain receptor 1 (DDR1)/ERK signaling. In turn, the suppression of collagen/DDR1 signaling by PGC1α leads to decreased levels of the known EMT regulators SNAIL1 and 2. Collectively, our results demonstrate a novel role for PGC1α in the regulation of proinvasive SNAIL proteins.

Developmental regulation of Th17-cell capacity in human neonates.

Human neonates are at significantly greater risk of serious infection than immunocompetent adults. In particular, very low birth weight infants in the neonatal intensive care nursery are at high risk of developing life-threatening bacterial and fungal infections. Recent studies have identified Th17 cells as critical mediators of immunity to bacterial and fungal infections at epithelial barriers. Little is known, however, about the ontogeny of Th17-cell responses in humans. The frequency of serious bacterial infections in preterm infants and the importance of Th17 cells in providing protection against such infections in animal studies prompted us to study Th17-cell development in human neonates. Naïve CD4(+) T cells from extremely preterm infants, term infants, and adults were assayed for their capacity to develop into Th17 effector cells. Surprisingly, Th17-cell capacity was inversely related to developmental age. Neonates expressed higher levels of IL-23R, RORγt, and STAT3 prior to activation and showed a significant Th17-cell bias after activation. In contrast, adult cells expressed more TBX21 with a corresponding Th1-cell bias. CD161 expression on Th17-cell precursors was also developmentally regulated. Our results suggest there is significant developmental regulation of CD4(+) effector lineages with a strong bias toward Th17-cell development early in life.

Preferences for the selection of unique tRNA primers revealed from analysis of HIV-1 replication in peripheral blood mononuclear cells.

BACKGROUND: All human immunodeficiency virus (HIV-1) uses a host tRNALys,3 as the primer for reverse transcription. The tRNALys,3 is bound to a region on the HIV-1 genome, the primer-binding site (PBS), that is complementary to the 18 terminal nucleotides of tRNALys,3. How HIV-1 selects the tRNA from the intracellular milieu is unresolved. RESULTS: HIV-1 tRNA primer selection has been investigated using viruses in which the primer-binding site (PBS) and a sequence within U5 were altered so as to be complementary to tRNAMet, tRNAPro or tRNAIle. Analysis of the replication of these viruses in human peripheral blood mononuclear cells (PBMC) revealed preferences for the selection of certain tRNAs. HIV-1 with the PBS altered to be complementary to tRNAMet, with and without the additional mutation in U5 to be complementary to the anticodon of tRNAMet, stably maintains the PBS complementary to tRNAMet following extended in vitro culture in PBMC. In contrast, viruses with either the PBS or PBS and U5 mutated to be complementary to tRNAIle were unstable during in vitro replication in PBMC and reverted to utilize tRNALys,3. Viruses with the PBS altered to be complementary to tRNAPro replicated in PBMC but reverted to use tRNALys,3; viruses with mutations in both the U5 and PBS complementary to tRNAPro maintained this PBS, yet replicated poorly in PBMC. CONCLUSION: The results of these studies demonstrate that HIV-1 has preferences for selection of certain tRNAs for high-level replication in PBMC.

Inhibition of human immunodeficiency virus type 1 replication by siRNA targeted to the highly conserved primer binding site.

The initiation of HIV-1 reverse transcription occurs at an 18-nucleotide sequence in the viral genome designated as the primer binding site (PBS), which is complementary to the 3' terminal nucleotides of tRNA(Lys,3). Since the PBS is highly conserved among all infectious HIV-1, it represents an attractive target for the development of new therapeutics to inhibit viral replication. In this study, we have evaluated three approaches using small interfering RNA (siRNAs) targeted to the PBS for the capacity to inhibit HIV-1 replication. In the first, transfection of a 21-nucleotide siRNA complementary to the PBS into cells inhibited production of HIV-1 following infection. Control siRNAs of the same length complementary to HIV-1 gag mRNA or to gfp mRNA decreased the production of virus or had no effect on virus replication, respectively. Analysis of the PBS of integrated proviruses derived from viruses that ultimately grew in cultures transfected with siRNA all contained wild-type PBS sequence, demonstrating that HIV-1 did not mutate to escape inhibition by siRNA. In the second approach, hairpin siRNA targeted to the wild-type PBS were expressed using an adeno-associated virus (AAV) vector. HIV-1 replication was inhibited in cells infected with AAV encoding the siRNA to the wild-type PBS, but not in cells infected with AAV encoding an siRNA of the same length targeted to an irrelevant PBS. Finally, studies from this laboratory have shown that alteration of the PBS to be complementary to tRNAHis results in the production of infectious virus that rapidly reverts to utilize tRNALys,3 following in vitro culture. A proviral genome containing a PBS complementary to tRNAHis that encodes an siRNA molecule complementary to the wild-type PBS under control of a U6 promoter within the nef gene was as infectious as the parent HIV-1 genome containing no insert in nef. The virus with the PBS only complementary to tRNAHis reverted to use tRNALys,3, coincident with rapid virus growth, while the virus encoding siRNA grew slower than the virus without siRNA and maintained the PBS complementary to tRNAHis longer in culture. At later times of infection, viruses with the PBS complementary to tRNAHis and the siRNA exhibited a rapid increase in p24 antigen in the culture. Analysis of the PBS revealed that it was now complementary to tRNALys,3. Analysis of the gene encoding the siRNA revealed that the reversion of the PBS coincided with the deletion of the gene encoding siRNA. The results of these studies show that siRNA targeted to the PBS of HIV-1 can inhibit virus replication, supporting the concept that HIV-1 has evolved a strong preference to select tRNALys,3 for high-level replication and establishing the PBS and primer selection as a potential target for new therapeutics.

HIV type 1 that select tRNA(His) or tRNA(Lys1,2) as primers for reverse transcription exhibit different infectivities in peripheral blood mononuclear cells.

The replication in human peripheral blood mononuclear cells (PBMC) of unique HIV-1 that select tRNA(His) or tRNA(Lys1,2) for reverse transcription was compared to the wild-type virus that uses tRNA(Lys,3). HIV-1 with only the primer-binding site (PBS) changed to be complementary to these alternative tRNAs initially replicated more slowly than the wild-type virus in PBMC, although all viruses eventually reached equivalent growth as measured by p24 antigen. Viruses with only a PBS complementary to the 3' terminal 18 nucleotides of tRNA(His) or tRNA(Lys1,2) reverted to use tRNA(Lys3). HIV-1 with mutations in the U5-PBS to allow selection of tRNA(His) and tRNA(Lys1,2) following long-term growth in SupT1 cells were also evaluated for growth and PBS stability following replication in PBMC. Although both viruses initially grew slower than wild type, they maintained a PBS complementary to the starting tRNA and did not revert to the wild-type PBS after long-term culture in PBMC. Analysis of the U5-PBS regions following long-term culture in PBMC also revealed few changes from the starting sequences. The virus that stably used tRNA(His) was less infectious than the wild type. In contrast, the virus that stably used tRNA(Lys1,2) evolved to be as infectious as wild-type virus following extended culture in PBMC. The results of these studies highlight the impact of the host cell on the tRNA primer selection process and subsequent infectivity of HIV-1.